Suitability of Collection Tubes with Separator Gels for Collecting and Storing Blood Samples for Therapeutic Drug Monitoring (TDM)

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1 Clin Chem Lab Med 2000; 38(4): by Walter de Gruyter Berlin New York Suitability of Collection Tubes with Separator Gels for Collecting and Storing Blood Samples for Therapeutic Drug Monitoring (TDM) Jouni Karppi 1, Kari K. Åkerman 2 and Markku Parviainen 1 1 Department of Clinical Chemistry, Kuopio University Hospital and University Kuopio, Finland 2 Laboratory Department, Kainuu Central Hospital, Kajaani, Finland In this study, we present significant changes occurring in serum drug concentrations while using blood collection tubes that contain a barrier gel. This report also contains results with antidepressant drugs, which have not been studied before with human samples. The drug concentrations were measured either with high performance liquid chromatography (HPLC) or fluorescence polarization immunoassay (FPIA). The results show that gel tubes are suitable for blood collection for antiepileptic, antibiotic, asthma and cardioactive drug measurements, since only slight adsorption was seen (0 5%). However, the studied tubes are not suitable for blood collection of antidepressants nor benzodiazepines, because the adsorption can be 5 30%. The adsorption was even higher (up to 40%) when samples were stored for 24 h after centrifugation in gel tubes. When the centrifugation step was performed after storage the effect of the barrier gel was lower (only 0 13%). Antidepressant drug measurements performed from patient specimens collected in the studied gel tubes and stored for 3 h showed <10% adsorption of the studied drugs. After 24 h storage time, concentrations of all analysed drugs decreased even more: adsorbed amount of drugs were about 5 20%. The studied gel tubes are proposed to be satisfactory for blood collection for antidepressant drug measurements if separation step is performed within 3 h after blood clotting. With the spiked samples the adsorption to barrier gel was higher, so it seems that adsorption is faster when drugs are not highly bound to serum proteins. Key words: Barrier gels; Serum separator blood collection tubes; Therapeutic drug monitoring (TDM). Abbreviations: FPIA, fluorescence polarization immunoassay; HPLC, high performance liquid chromatography; LD, lactate dehydrogenase. Introduction Blood collection tubes containing gel barriers have gained widespread acceptance due to advantages of barrier gel including less manipulation required in processing specimens collected in these tubes, and they minimize exposure of laboratory workers to infectious hazards. Serum yield obtained from gel tubes is increased and staff time in processing is reduced (1). The most often used gel types are acryls, polyesters and silicones. Serum separator blood collection tubes are coated with a clot activator that is usually silicone oxide (2). The effect of barrier gels on analyte concentrations has been studied in several investigations (3, 4). Already early in the 1970 s (3) it was proposed that gelcontaining tubes would be fast and efficient for serum separation from blood cells. Thirty four clinical chemistry analytes were evaluated and the results only differed for urea, lactate dehydrogenase (LD), LD2 and CO 2. Drug measurements were made only for lithium and salicylate, and there were no major differences noted in their concentrations. Chan et al. (4) have also investigated the suitability of gel barrier-containing tubes on blood sampling for clinical chemistry. They reported that the effect was only minor, and the tubes would be suitable for routine use. The suitability of gel tubes for blood sampling for therapeutic drug monitoring has been investigated in some publications (5 7). Reports describe antiepileptic, antibiotic and cardioactive drug concentrations analyzed from samples obtained with gel tubes. The results show that drug concentrations in samples obtained with gel tubes are often lower than the corresponding samples in plain tubes. This could be explained by adsorption of drugs onto the gel material. It has been pointed out that a polyester based gel could adsorb many drugs such as antiepileptics (5). Storage time and sample volume will also contribute to the observed drug concentration. The suitability of Vacutainer SST (Becton Dickinson, Vacutainer Systems Eur, Meylan, France), Autosep (Terumo, Europe N.V, Leuven, Belgium) and Microvette (Sarstedt, Rommeldorf, Germany) gel tubes for antiepileptic drug measurements was investigated by Berqvist et al. (5). They observed that concentrations of phenobarbital and carbamazepine were lower in samples obtained in gel tubes when samples were stored for 24 h. The longer the drug was in the gel tube, the lower the observed concentration was. After a one week storage time, the concentration of phenytoin was also lower in Vacutainer SST gel tubes than in plain tubes. Dasgupta et al. have studied the stability of seven of the most commonly measured drugs in Vacutainer SST and Corvac gel tubes (6, 7). They observed that there was a marked decrease in concentration when phenytoin, phenobarbital, carbamazepine, quinidine and lidocaine samples were stored for h. This decrease in the concentrations was more marked

2 314 Karppi et al.: Gel-containing blood collection tubes when sample volume was as low as µl. Larger samples, for example 1 ml, had less effect on the concentration decrease. The gel used in the Corvac gel tube was quite inert to most drugs. They observed no effect of long storage time and small sample volume on the concentration difference between Corvac gel tubes and plain tubes. Landt et al. (2) studied the suitability of three kinds of gel-containing sampling tubes with different gel materials on blood collection for drug measurements (acryl, silicone and polyester polymers). None of them had any significant effect on the measured drug concentrations (theophylline, digoxin, phenytoin, phenobarbital, gentamycin, ethanol and cyclosporin), when samples were prepared and analysed immediately after blood clotting. In this report we have evaluated the suitability of three different blood sampling tubes containing gel barriers (Vacutainer SST, Vacuette and Venoject II (Autosep)) for therapeutic drug monitoring (TDM). We studied the effect of prolonged storage time for the drug concentrations measured from both spiked and authentic patient serum specimens. This report contains also results on many antidepressant drugs which have not been investigated previously. (5 ml) and Venoject plain tubes (10 ml) were obtained from Terumo Europe N.V (Leuven, Belgium). Apparatus The concentrations of antibiotic, asthma, and cardioactive drugs were analyzed with Abbott AxSYM (Abbott Laboratories Diagnostics Division, Irving, TX, USA) or TDx (Abbott Laboratories, Irving, TX, USA) analyzer. Other drugs were analyzed by HPLC with methods described by Åkerman et al. (8 11) or with other slightly modified methods. The impresicion data of HPLC methods is illustrated in Table 1. Briefly, the samples and standards were prepared with an automatic sample preparator (Gilson Medical Electronics, Villiers-le-Bel, France) or manual sample preparation using 100 mg Bond-Elut C 18 solid-phase extraction columns (Varian, Sunnyvale, CA, USA). Chromatographic separations were achieved by using a Select-B C 8 4 x 125 mm column (Merck), Symmetry C x 150 cm column (WATERS), LiChroCART C x 250 mm column (Merck) or NovaPak C x 150 mm column. The elution was isocratic with a mobile phase of acetonitrile-10 mm dipotassium hydrogen phosphate, ph 4.7 (40:60) at a flow-rate of 1.2 ml/min or with a mobile phase of methanol-acetonitrile-50 mm dipotassium hydrogen phosphate, ph 4.7 (2:30:68) at a flow-rate of 1.5 ml/min. The drugs were detected at 220, 240 or 257 nm and peak purity analyses were performed at nm. A Hewlett Packard Series 1050 liquid chromatography system Materials and Methods Reagents Alprazolam, mianserin, perphenazine, phenobarbital and phenytom were purchased from Orion (Helsinki, Finland). Amitriptyline, citalopram, desmethylcitalopram and nortriptyline were obtained from H.Lundbeck A/S (Copenhagen-Valby, Denmark). Clobazam and norclobazam were obtained from Hoechst (Frankfürt/Main, Germany). Clomipramine, desipramine, hydroxycarbazepine, imipramine, norclomipramine, oxcarbazepine, protriptyline were purchased from Ciba-Geigy (Basel, Switzerland). Clonazepam and flunitrazepam were from Roche (Basel, Switzerland). Clozapine and norclozapine were obtained from Sandoz (Berne, Switzerland). Diazepam, nordiazepam and oxazepam were obtained from Dumex (Copenhagen, Denmark). Doxepin and nordoxepin were obtained from Pfizer (Brussels, Belgium). Fluoxetine and norfluoxetine were from Lilly (Indianapolis, USA). Lamotrigine was obtained from the Wellcome Foundation (London, England). Nitrazepam was obtained from Leiras (Turku, Finland). Primidone was obtained from Cambridge Research Biochemicals (Chesire, UK). Trimipramine and nortrimipramine were from Rhone-Poulenc Rorer (Birkenrod, Denmark). Vigabatrin (GVG) was purchased from Astra (Södertälje, Sweden). High performance liquid chromatography (HPLC)-grade acetonitrile and methanoi as well as other analytical grade reagents were obtained from Merck (Darmstadt, Germany). Blood collection tubes Vacutainer SST serum separator and red top plain (5 ml) blood collection tubes were obtained from Becton-Dickinson (Vacutainer Systems Eur, Meylan, France), Vacuette serum separator and plain blood collection tubes (5 ml) were purchased from Greiner Labortechnic (Kremsmünster, Austria), Venoject II (Autosep) serum separator and plain blood collection tubes Tab. 1 The imprecision data of the HPLC methods. Drug Intra-asay Inter-assay Alprazolam Carbamazepine Citalopram Clobazam Clomipramine Clonazepam Clozapine Desipramine Desmethylcitalopram Desmethylclomipramine Diazepam Doxepin Fluoxetine Hydoxycarbazepine <5 <5 Imipramine Lamotrigine Mianserin Nitrazepam Norclobazam Norclozapine Nordiazepam Nordoxepin Norfluoxetine Nortrimipramine Oxazepam Oxcarbazepine <5 <5 Perphenazine Phenobarbital <5 <5 Phenytoin <5 <5 Primidone Trimipramine Vigabatrin (GVG)

3 Karppi et al.: Gel-containing blood collection tubes 315 (HP Series 1050 sampler, HP Series 1050 Quaternary pump, HP Series 1050 Diode Array Detector) controlled by chemstation no. Aa (Palo Alto, CA, USA), a Hewlett Packard Series 1100 liquid chromatography system (HP Series 1100 sampler, HP Series 1100 Quaternary pump, HP Series 1100 Diode Array Detector) controlled by Chemstation no. Aa (Palo Alto, CA, USA) and a Perkin-Elmer liquid chromatography system (ISS 200 autosampler, Binary LC 250 pump, 235C diode-array detector), controlled by Turbochrom chromatography workstation (Perkin-Elmer, Norvalk, CT, USA), were used. Sample collection Blood samples for determination of antiepileptic and antidepressant drug concentrations were collected from free volunteers into five (10 ml) plain tubes (Venoject). Stock solutions of each drug were prepared by dissolving 10 mg of each in 10 ml of methanol in separate bottles (1 mg/ml). The final drug solution was made by diluting appropriate amounts of stock solutions with 5% methanol-water solution. A 100 µl portion of that drug solution was transferred to the plain blood collection tube before filling with blood. The concentration of each drug was calculated to be in the therapeutic range. After mixing, the volume of 4 ml of blood was divided into both gel and plain tube (V=5 ml). After blood clotting, the samples were centrifuged at approximately 1800 g for 10 min, the serum was promptly separated and stored at +4 C until analysis within one week (zero time samples). Another series of serum samples was stored after centrifugation in gel tubes for 24 h at +4 C. The third sample series was stored without centrifugation in gel tubes for 24 h at +4 C, followed by centrifugation and serum separation after that period. Serum specimens containing antibiotics, cardioactive drugs, asthma drugs, valproic acid and carbamazepine were collected in Vacutainer SST gel tubes from patients taking these drugs, and the drug analysis was made immediately after blood clotting and centrifugation. In addition, the serum samples containing antidepressants and benzodiazepines stored for 3 and 24 h in gel tubes were pooled from patients receiving these medications to study the stability of drug concentrations. From this serum pool, 2.5 ml of serum was transferred to each gel and plain tube to be studied in this research. Statistical analysis Drug concentration in the gel tube was compared with the plain tube to obtain the recovery in the gel tube. From these recovery averages, standard deviations (SD) and coefficients of variation (CV) were calculated. The differences of drug concentrations between investigated gel and plain tubes were calculated with the Microcal Origin software (One Roundhouse Plaza, Northampton, MA, USA), using two-tailed paired t-test at the significance level 0.05 (version 4.1, 16 bit.). The drug recovery obtained from zero time samples was compared with samples of serum or whole blood stored for 24 h to follow recoveries between sample series. Results Vacutainer SST gel tube comparison with Vacutainer plain tube In samples analysed immediately after blood clotting the recoveries of tricyclic antidepressants and the other psychoactive drugs varied from 73.2 to 95% in gel tubes and the adsorption was statistically significant for nearly all the antidepressants. The adsorption of clonazepam, nitrazepam and alprazolam (recoveries <93%) was also significant. Only slight concentration differences were observed for diazepam and its metabolites between gel and plain tubes. For the studied antiepileptics, a minimal difference of drug concentrations between plain and gel tubes was noted. For carbamazepine, the recovery was relatively low (93.6%). The concentrations of theophylline, digoxin and antibiotics (patient samples) remained stable in the gel tube when serum was separated and analysed promptly (recoveries varied from 97.5 to 103.2%) (Table 2). Recoveries of tricyclic antidepressants, clozapine, diazepam and antiepileptics decreased even more after 24 h storage interval as serum (recovery range from 67.7 to 96.2%) compared with the zero time samples (Table 3). Neither hydroxycarbazepine nor lamotrigine were adsorbed to the gel material to any significant extent. In samples stored for 24 h without centrifugation, all recoveries were >90% (Table 4). Vacuette gel tube comparison with Vacuette plain tube In samples separated immediately after blood clotting, the concentrations of tricyclic antidepressants decreased significantly (except of nordoxepine) in the gel tube (recoveries varied from 72.7 to 88.6%). The concentrations of other antidepressants, clozapine and diazepam were lower in the gel tube and the mean level of adsorption was also statistically significant (recoveries were from 86.9 to 96.4%). The concentrations of hydroxycarbazepine, lamotrigine and phenobarbital did not change significantly in the gel tube (Table 2). After storage for 24 h, the adsorption rate was higher than in samples that were analysed immediately after blood clotting (Table 3). The adsorbed amounts of drugs obtained from whole blood samples stored for 24 h were much lower than in serum samples not stored or stored for 24 h (Table 4). Venoject II (Autosep) gel tube comparison with Venoject II plain tube The adsorbed amounts of tricyclic antidepressants (except of nortriptyline and nordoxepine), clozapine and diazepam were observed to be statistically significant (recoveries varied from 81.8 to 94.6%) in samples analysed immediately after blood clotting and centrifugation. In contrast, hydroxycarbazepine, lamotrigine and phenobarbital concentrations did not decrease significantly under identical conditions (Table 2). The adsorption of tricyclic antidepressants in serum samples stored for 24 h was noted to be statistically significant, although recovery of nortriptyline was as high as 97%. No adsorption was detected for norfluoxetine, diazepam, hydroxycarbazepine, lamotrigine and phenobarbital. Clozapine adsorbed to quite a large extent to the gel material (recovery 86.8%) (Table 3). The adsorption of amitriptyline to the gel material was marked in the whole blood samples stored for

4 316 Karppi et al.: Gel-containing blood collection tubes Tab. 2 Drug recovery from the zero time serum samples in gel tubes compared with the plain tubes. Drug Concentration N Recovery (%) ± SD (%) p-value Recovery (%) ± SD (%) p-value Recovery (%) ± S.D.% p-value Amitriptyline 260 nmol/l ± 2.1 * 6.1E ± 3.4 * ± 4.4 * 0.02 Clomipramine 750 nmol/l ± Desipramine 300 nmol/l ± 2.0 * 2.2E-4 Desmethylclomipramine 790 nmol/l ± 8, Doxepin 320 nmol/l ± 3.5 * 2.9E ± 2.0 * 4.0E ± 11.4 * 0.03 Imipramine 690 nmol/l ± 2.2 * 9.2E-5 Nordoxepin 200 nmol/l ± 4.2 * ± ± Nortrimipramine 160 nmol/l ± 8.7 * 0.08 Nortriptyline 140 nmol/l ± 1.8 * 8.0E ± 3.0 * 3.3E ± Trimipramine 220 nmol/l ± 2.5 * 2.4E-4 Citalopram 360 nmol/l ± 6.2 * Desmethylcitalopram 130 nmol/l ± Fluoxetine 860 nmol/l ± 4.4 * ± ± Mianserin 250 nmol/l ± 3.5 * Norfluoxetine 750 nmol/l ± 3.2* ± 8.5 * ± Perphenazine 3.7 nmol/l ± Clozapine 1700 nmol/l ± 8.0 * ± ± 4.4 * 0.02 Norclozapine 2530 nmol/l ± 14.1 * ± ± Alprazolam 550 nmol/l ± 1.1 * 1.77E-4 Clonazepam 380 nmol/l ± 1.2 * 1.06E-5 Diazepam 1.8 µmol/l ± ± ± 2.6 * 0.02 Nitrazepam 880 nmol/l ± 10.7* 4.3E-5 Nordiazepam 3.4 µmol/l ± Oxazepam 4.7 µmol/l ± Carbamazepine 30 µmol/l ± 6.5 * 5.5E-6 Clobazam 780 nmol/l ± 2.7 * 0.03 Ethosuximide 40 µmol/l ± Hydoxycarbazepine 110 µmol/l ± 1.6* ± 2.0 * ± Lamotrigine 4.5 µmol/l ± ± ± Norclobazam 440 nmol/l ± Oxcarbazepine 1.5 µmol/l ± Phenobarbital 30 µmol/l ± ± ± Phenytoin 50 µmol/l ± Primidone 13 µmol/l ± Valproic acid 360 µmol/l ± Vigabatrin (GVG) 180 µmol/l ± Asthma drugs Theophylline 30 µmol/l ± Aminoglycosides Netilmycin 1.3 mg/l ± Tobramycin 2.4 mg/l ± Other antibiotics Vancomycin 16 mg/l ± Cardioactive drugs Digoxin 1.3 nmol/l ± * = statistically significant deviation at the 0.05 level This study was carried out with added drugs except for carbamazepine, ethosuximide, phenytoin, valproic acid, theophylline, aminoglycosides, vancomycin and digoxin that were measured from patient samples. 24h. No adsorption of other drugs was detected, and their concentrations remained relatively stable in the gel tube (Table 4). Authentic patient serum samples Since the spiked samples are not identical to patient samples (e.g. protein binding) the adsorption levels of drugs were also measured with authentic patient sam-

5 Karppi et al.: Gel-containing blood collection tubes 317 Tab. 3 Drug recovery from the serum samples stored for 24 h in gel tubes compared with the plain tubes at +4 C. Drug N Recovery (%) ± SD (%) p-value Recovery (%) ± SD (%) p-value Recovery (%) ± SD (%) p-value Amitriptyline ± 3.5 * 1.2E ± 2.9 * 3.2E ± 1.1 * 6.8E-6 Nortriptyline ± 3.0 * 2.2E ± 2.5 * 4.3E ± 1.6 * 0.02 Doxepin ± 2.5 * 4.1E ± 4.7 * 3.1E ± 4.2 * 6.2E-4 Nordoxepin ± 1.5 * 5.0E ± ± Fluoxetine ± 5.1 * ± 4.8 * ± Norfluoxetine ± 3.6 * ± ± Clozapine ± 3.2 * 2.3E ± 3.8 * 5.3E ± 6.0 * 0.01 Norclozapine ± 5.5 * ± 5.2 * ± Diazepam ± 2.9 * ± 3.4 * ± Hydroxycarbazepine ± ± ± Lamotrigine ± ± ± Phenobarbital ± ± ± * = statistically significant deviation at the 0.05 level This study was carried out with added drugs. Tab. 4 Drug recovery from the whole blood samples stored for 24 h in gel tubes compared with the plain tubes at +4 C. Drug N Recovery (%) ± SD (%) p-value Recovery (%) ± SD (%) p-value Recovery (%) ± SD (%) p-value Amitriptyline ± ± 2.0 * 5.9E ± Nortriptyline ± 1.4 * 7.0E ± 3.5 * ± 6.1 * 0.03 Doxepin ± ± 2.8 * ± Nordoxepin ± 0.9 * 8.5E ± ± Fluoxetine ± ± ± Norfluoxetine ± ± ± Clozapine ± 1.2 * 2.6E ± ± Norclozapine ± 1.7 * 8.8E ± 3.9 * ± 3.3 * Diazepam ± ± ± Hydroxycarbazepine ± ± ± Lamotrigine ± ± ± Phenobarbital ± ± ± * = statistically significant deviation at the 0.05 level This study was carried out with added drugs. ples. Drug concentrations measured from patient serum samples collected in Vacutainer SST, Vacuette and Venoject II (Autosep) gel tubes and stored for 3 h showed no statistically significant adsorption of antiepileptics (exept of clonazepam) and antidepressants. It was observed that the adsorption of clonazepam was marked, and its concentration did not remain stable after storing serum for 3 and 24 h in all tested gel tubes, potentially causing errors for patient drug treatment. Surprisingly, concentrations of antidepressants remained relatively stable in gel tubes after 3h storage interval (adsorbed amount of studied drugs only <10%). After one day storage period, concentrations of all antidepressants decreased even more in all gel tubes, and adsorption was noted to be marked. Adsorption was statistically significant for amitriptyline, nortriptyline (Vacutainer SST and Vacuette), nordoxepin (Vacuette and Venoject II Autosep)

6 318 Karppi et al.: Gel-containing blood collection tubes and norfluoxetine (Venoject II Autosep). Concentrations of fluoxetine remained quite stable in gel tubes. The concentrations of clozapine and its metabolite desmethylclozapine decreased slightly in samples stored for 3 h in Vacutainer SST gel tubes (adsorbed amounts were about 6%). Concentrations of drugs decreased even more in specimens stored for 24 h (Tables 5 and 6). Discussion Antiepileptic drugs (except for carbamazepine), antibiotics, digoxin, theophylline, diazepam and its metabolites are able to be analyzed from blood samples collected in Vacutainer SST gel tubes containing polyester-based polymer as gel material immediately after blood clotting and centrifugation, since their concentrations stayed relatively stable in the gel tube and the decrease of drug concentrations was not clinically significant. There are some previous studies about evaluation of Vacutainer SST gel tubes for blood collection of TDM. Berqvist et al. (5) and Dasgupta et al. (6) also evaluated suitability of Vacutainer SST gel tube for blood collection for antiepileptic drug measurement and they observed that concentrations of phenytoin, carbamazepine and phenobarbital decreased significantly after 24 h storage interval in the gel tube. Quattrocchi et al. (12) reported loss of phenytoin, lidocaine and phenobarbital. Clave et al. (13) claimed the SST tube to be satisfactory for phenobarbital, theophylline, tobramycin, and digoxin. Bailey et al. (14) found no effect of the Corvac tube on 11 drugs. Some other authors have studied other drugs or tubes. The SST tube was not found to be satisfactory for tricyclic antidepressants (15). In this study, the concentration of phenobarbital was observed to be relatively stable after storage interval, and this study also confirms suitability of SST tubes for drug measurement described by other authors except for antidepressants. Concentrations of antidepressants and benzodiazepines decreased significantly in Vacutainer SST gel tube. When collecting samples from patients receiving these medications, the decrease of drug concentrations was not as dramatic, except for clonazepam after 3 h storage interval. Concentrations of antidepressants and neuroleptics decreased even more after one day storage time, and therefore it is proposed that Vacutainer SST blood collection tubes are suitable for blood collection for antidepressant drug measurements if separation step is performed within 3 h. Drug recoveries were relatively similar comparing serum specimens either measured without storage or stored for 24 h. Specimens stored for 24 h without centrifugation showed the recoveries of all studied drugs to be >90%. Vacuette gel tubes containing unhalogenated olefin oligomer as gel material are satisfactory only for blood collection for antiepileptic drug measurements. However, in blood collected from patients taking these drugs the concentrations of antidepressants did also remain relatively stable after 3 h storage period in the gel tube. After one day storage time, the concentrations decreased even more, as in Vacutainer SST gel tubes, and therefore the separation step should be done within 3 h, since decrease of drug concentrations was not statistically significant. Recoveries of drugs were much better in samples stored for 24 h without centrifugation compared with samples without storage or stored as serum 24 h (recoveries >86%). Venoject II (Autosep) gel tubes containing thixotropic synthetic polymer as gel material are proposed to be suitable for blood collection for antiepileptic drug and diazepam measurements. This gel material was noted to be relatively inert for those drugs. Some other authors have also tested suitability of Venoject II (Autosep) gel tube for blood collection for antiepileptic drug mea- Tab. 5 Drug recovery from the patient serum samples stored for 3 h in gel tubes compared with the plain tubes at +4 C. Drug Concentration N Recovery (%) ± SD (%) p-value Recovery (%) ± SD (%) p-value Recovery (%) ± SD (%) p-value Amitriptyline 83 nmol/l ± ± ± Doxepin 34 nmol/l ± ± ± Nordoxepin 35 nmol/l ± ± ± Nortriptyline 64 nmol/l ± ± ± Fluoxetine 470 nmol/l ± ± ± Norfluoxetine 750 nmol/l ± ± ± Clozapine 1720 nmol/l ± ± ± Norclozapine 780 nmol/l ± ± ± Clonazepam 94 nmol/l ± ± ± Hydroxycarbazepine 72 µmol/l ± ± ± Lamotrigine 14 µmol/l ± ± ±

7 Karppi et al.: Gel-containing blood collection tubes 319 Tab. 6 Drug recovery from the patient serum samples stored for 24 h in gel tubes compared with the plain tubes at +4 C. Drug Concentration N Recovery (%) ± SD (%) p-value Recovery (%) ± SD (%) p-value Recovery (%) ± SD (%) p-value Amitriptyline 54 nmol/l ± ± 1.0 * 5.0E ± Doxepin 170 nmol/l ± ± ± Nordoxepin 24 nmol/l ± ± 3.4 * ± 1.8 * Nortriptyline 62 nmol/l ± 2.1 * ± 1.4 * ± Fluoxetine 450 nmol/l ± ± ± Norfluoxetine 390 nmol/l ± ± ± 0.8 * Clozapine 1600 nmol/l ± ± ± Norclozapine 780 nmol/l ± ± ± Clonazepam 94 nmol/l ± 0.9 * ± 4.4 * ± Hydroxycarbazepine 80 µmol/l ± ± ± Lamotrigine 12 µmol/l ± ± ± * = statistically significant deviation at the 0.05 level surement. Berqvist et al. (5) and Dasgupta et al. (6) also evaluated suitability of Venoject (Autosep) gel tube for blood collection for antiepileptic drug measurement and they noted a loss of phenytoin, carbamazepine and phenobarbital concentrations after 24 h storage interval in the gel tube. This study showed no significant decrease of phenobarbital concentration. Concentrations of antidepressants decreased dramatically if the centrifugation step was performed immediately after blood coagulation and samples were stored in contact with barrier gel for 24 h. There was no statistically significant adsorption for the antidepressants in the authentic patient samples. Rate of adsorption increased after 24 h storage interval. Venoject II (Autosep) gel tubes seem to be suitable for blood collection for antidepressant drug measurements when separation step is performed within 3 h. It was observed that decrease of drug concentrations in the gel tube was diminished when specimens were stored for 24 h without centrifugation. Conclusions Recovery of the studied drugs obtained from all investigated blood collection tubes containing barrier gels was similar. Studied gel materials were relatively inert for antiepileptic drugs, but spiked concentrations of antidepressants and benzodiazepines decreased dramatically in these gel tubes without storage or stored for 24h as serum. Spiked specimens stored for 24h as whole blood showed drug recovery of about %, and it was proposed the presence of erythrocytes may cause a smaller decrease of drug concentrations during storage period. These gel tubes were observed to be suitable for blood collection for analysis of antiepileptic drugs, asthma drugs (theophylline), antibiotics and cardioactive drugs (digoxin) (zero time) investigated in this study. These gel tubes were not suitable for blood collection for antidepressant drug measurements. The recovery of antidepressant drugs was near 100% in patient samples stored in gel tubes after centrifugation at 3 h. With spiked samples the recovery was remarkably low and it seems that the adsorption is due to protein binding. This claim must be clearly investigated before these tubes could be used in sample collection for TDM. In many laboratories the samples are collected in separated places and therefore stored several hours in blood collection tubes. This report shows clearly that, for TDM purposes, the samples must be taken in ordinary serum tubes. In all cases the concentrations of antidepressants and benzodiazepines are decreased dramatically during 24 h storage. Acknowledgements The authors thank personnel of the laboratory for technical assistance. References 1. Koch TR, Platoff G. Suitability of collection tubes with separator gels for therapeutic drug monitoring. Ther Drug Monit 1990; 12: Landt M, Smith CH, Hortin GL. Evaluation of evacuated blood collection tubes: effects of three kind types of polymeric separators on therapeutic drug-monitoring specimens. Clin Chem 1993; 39: Mathies JC. Evaluation of a new device for rapidly separating serum or plasma from blood. Clin Chem 1974; 20: Chan K-M, Daft M, Koenig JW, Ladenson JH. Plasma separator tube of Becton Dickinson evaluated. Clin Chem 1988; 10:

8 320 Karppi et al.: Gel-containing blood collection tubes 5. Bergqvist Y, Eckerblom S, Fundig L. Effect of use of gelbarrier sampling tubes on determination of some antiepileptic drugs in serum. Clin Chem 1984; 30: Dasgupta A, Dean R, Saldana S, Kinnaman G, McWhon RW. Absorption of therapeutic drugs by barrier gels in serum separator blood collection tubes. Am J Clin Path 1994; 101: Dasgupta A, Blackwell W, Bard D. Stability of therapeutic drug measurement in Vacutainer plastic blood-collection tubes. Ther Drug Monit 1996; 18: Åkerman KK, Jolkkonen J, Parviainen M, Penttilä I. Analysis of low-dose benzodiazepines by HPLC with automated solid-phase extraction. Clin Chem 1996; 42: Åkerman KK. Analysis of clobazam and its active metabolite norclobazam in plasma and serum using HPLC/DAD. Scand J Clin Lab Invest 1996; 56: Åkerman KK. Analysis of clozapine and norclozapine by high-performance liquid chromatocraphy. J Chromatogr B 1997; 696: Åkerman KK, Jolkkonen J, Huttunen H, Penttilä I. High-performance liquid chromatography method for analysing citalopram and desmethylcitalopram from human serum. Ther Drug Monit 1998; 20(1): Qattrocchi R, Karnes HT, Robinson D, Hendeles L. Effect of serum separator blood collection tubes on drug concentrations. Ther Drug Monit 1983; 5: Clave LP, Beltran LG, Liuch MP, Mostaza CP, Ricra SS. Blood collection tubes evaluated for use in measurement of some therapeutic drugs. Clin Chem 1988; 34: Bailey DN, Coffee JJ, Briggs JR. Stability of drug concentrations in plasma stored in serum separator blood collection tubes. Ther Drug Monit 1988; 10: Nyberg G, Martensson E. Preparation of serum and plasma samples for determination of tricyclic antidepressants: effects of blood collection tubes and storage. Ther Drug Monit 1986; 8: Received: 9 August 1999; accepted 22 December 1999 Corresponding author: Jouni Karppi, Department of Clinical Chemistry, Kuopio University Hospital and University, P.O.Box 1777, FIN Kuopio, Finland Tel.: , Fax.: kari.akerman@kass.fi

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