4. FULLY AUTOMATED PRETREATMENT OF BIOLOGICAL
4.1. Fully Automated Sample Pretreatment Systems
Dialysis is a barrier separation technique, in addition to osmosis and ultrafiltra- tion, among others. Mass transfer takes place through a semipermeable membrane located between two liquid (donor and acceptor) phases that need not be immisci- ble. Separations are kinetically controlled and based on the occurrence of concen- tration gradients between the two liquid phases, isolated by a selective membrane that discriminates solutes according to pore size and/or electric charge [39]. The applications of continuous dialysis as a purification step for biological fluids prior to GC–MS analysis has received little attention despite its significance to routine
256 Valca´rcel et al.
clinical laboratories. In addition, this technique has limited capability to imple- ment preconcentration (normally it is employed for sample dilution and inter- ferent removal), so continuous dialysis systems for drug analysis often include trace enrichment of the dialysate. Only off-line approaches including autosam- plers with sample preparation functions for liquid samples before injection into the GC have been used; thus, the automated sequential trace enrichment of dialy- sates (ASTED) instruments from Gilson have been employed for dialysis of bio- logical fluids. The earliest attempt in this context was made by Krogh et al. [15]
in the determination of opiates; the ASTED system used is schematically depicted in Figure 3. The dialysis unit was of the sandwich type and furnished with a microporous cuprophane membrane. The sample (blood) was held static in the donor channel of the dialyzer while 5 ml of 10⫺4M ammonia was delivered in pulses of 650àl; ammonia diffused through the membrane and into the sample,
Figure 3 Automated ASTED system for the determination of opiates in human blood.
C⫽polystyrene–divinylbenzene column; W⫽waste. See text for details.
Drug Determinations in Biological Fluids 257
which resulted in reduction of the protein-opiate binding without protein precipi- tation. Drug recoveries ranged from 66 to 80% for 9 minutes of dialysis. The dialysate in ammonia was preconcentrated on a SPE unit including a polystyrene–
divinylbenzene column (1 cm⫻2 mm); acetonitrile was used as eluent, and the first 100àl of eluate was discarded and the next 700àl was collected in glass vials. The eluates were evaporated and then derivatized. Approximately 100 sam- ples can thus be automatically prepared in 24 hr. The analytical features of this approach are shown in Table 2. A similar dialysis–SPE unit was used by Herra´ez- Herna´ndez et al. [16] for the determination of drugs in plasma, using some benzo- diazepines as model compounds. In contrast to the above-described method, no further manual steps were required after elution as no derivatization of the benzo- diazepines was necessary. The ASTED system included a similar dialysis unit with the same membrane [15], but the donor and acceptor channel volumes of the dialysis cell were lower (100 and 170àl, respectively) and both liquid (donor and acceptor) phases were water. For preconcentration, a PLRP-5 column was used and the analytes were eluted with 275àl of ethyl acetate. On-line experi- ments were done with FID and nitrogen–phosphorus detector (NPD) detection (linear range 0.1 to 20àg/ml benzodiazepines; relative standard deviation (RSD) 4 to 16%. With MS, 100-àl volumes of eluate were manually injected; this was only used for confirmation, so no analytical features were given.
Two commercially available automated devices for the preparation of bio- logical fluids based on SPE have been reported. One uses an HP-PrepStation (Hewlett-Packard, Palo Alto, CA) for the extraction and derivatization of narcot- ics in serum samples after enzymatic hydrolysis [11]; C18 cartridges were used for SPE. The other is a fully automated procedure applied to the confirmation by GC–MS of positives of cocaine and benzoylecgonine in previously screened urines, using a laboratory robotic system [22].
Continuous flow systems for the automated pretreatment of biological flu- ids for GC–MS of drugs have seemingly been developed by the authors’ group only. A CFS relying on SPE constitutes the most simple, robust, cheap, and fruit- ful approach in this context. Sample, reagents, and eluents are introduced into a continuous module furnished with a sorbent column located in the loop of an injection valve. The CFS operation comprises three steps: sorption (sample intro- duction), elution, and derivatization—the last two can occur simultaneously, however. The final extract obtained can be introduced into the GC–MS (Fig. 4).
The simplest version of such CFS was that developed for the rapid SPE–
derivatization, with volume-based sampling (1 ml of urine), of various abuse drugs (barbiturates, opiates, and cocaine, among others) in real human urine [19].
The sorbent material was polymeric Amberlite XAD-2 and there were no restric- tions on the retention pH as all the drugs assayed exhibited maximum adsorption within the range 6 to 9, which is within the normal urine pH range. The derivatis- ing reagent for silylation, BSTFA, was added to the eluent (trichloromethane
258Valca´rceletal.
Table 2 Applications of Automated Sample Pretreatment Systems to Drug Analyses by GC–MS (EI Mode) Separation principle/
Drug Matrix System derivatizing reagent Linear range (ng/ml) RSD (%) Ref.
Opiates Plasma and whole ASTED Dialysis and SPE/ 0.2–5.0 nmol/ml 1.3–7.7 15
blood BSTFAa
Benzodiazepines Plasma ASTED Dialysis and SPE Not stated Not stated 16
Narcotics Serum HP-PrepStation SPE/derivatization 2–250 0.5–2.0 11
Cocaine and benzoylecgonine Urine Zymate Robot SPE/derivatization 5–100 1.1–6.0 22
Abuse drugs Urine CFS SPE/BSTFA 1–2000 2.1–6.8 19
Antiinflammatory drugs Urine and plasmab CFS SPE/CH3I-K2CO3 50–3000 3.4–6.0 20 (horse)
Cocaine and its metabolites Urine CFS SPE/BSA 5–3000 3.1–5.8 21
Benzodiazepines Urinec CFS SPE 5–2000 4.5–6.5 42
Abuse drugs Urine and serum CFS SPE 5–2500 2.9–4.6 43
Caffeine Urine ASPEC SPE Not stated Not stated 44
aManual derivatization withN,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA).
bPlasma sample required manual liquid–liquid extraction before SPE.
cUrine was hydrolyzed with 3 M HCl (100°C, 1 hr).
Abbreviations: ASTED, automated sequential trace enrichment of dialysate; CFS, continuous flow system; ASPEC, automated sample preparation with extraction columns; SPE, solid phase extraction; BSA, bis(trimethylsilyl)acetamide.
Drug Determinations in Biological Fluids 259
Figure 4 Configurations of an SPE-based CFS for the automated pretreatment of biolog- ical fluids. IV⫽injection valve; W⫽waste. See text for details.
(CHCl3):acetone, 1 : 1, v/v) in a 1 : 1 ratio; the derivatization was carried out with- out halting the flow and at room temperature. One potential disadvantage of the method may be its unsuitability for on-line hydrolysis of the glucuronide conju- gates of the drugs, as the enzymatic reaction requires a long time (over 60 min- utes). However, the use of nonhydrolyzed urine allows one to monitor relevant metabolites (e.g., 6-O-monoacetyl morphine (6-MAM) from heroin, which would otherwise be decomposed [40] and whose presence is decisive for stating heroin use). Thus, as can be seen in Figure 5, codeine, morphine, and 6-MAM were detected in a heroin-user urine sample following automated sample pretreatment in the system of Figure 4.
An automated system for the SPE of 17 NSAIDs and on-line methylation prior to their off-line introduction into a GC–MS has also been proposed [20].
The ensuing method involves derivatization with methyl iodide (CH3I) in the presence of solid potassium carbonate (K2CO3) as catalyst (official method) and was satisfactorily applied to the determination of these drugs in racehorse urine and plasma. Amberlite XAD-2 was found to be the most convenient sorbent and acetonitrile the best eluent. No pH restrictions regarding NSAIDs were observed as they were completely retained within the range 5.5 to 10; however, elution required conditioning with dilute acetic acid. The major problem was the water traces remaining in the CFS, which hindered development of the derivatization reaction through dissolution of K2CO3. Acetic anhydride (20%, v/v) was added to the eluent to act both as drying agent and conditioner for the resin at an appro-
260 Valca´rcel et al.
Figure 5 EI mass spectra for opiates detected in a urine sample from a heroin addict.
See text for details.
priate pH. As in the previous system, CH3I (the derivatising reagent) was added to the eluent (25% v/v). The main technical disadvantage of this method results from compaction of the K2CO3 column (located after the sorbent column) but was overcome by packing the catalyst with sodium aluminosilicate pellets placed at intervals in the column. Under these conditions, the derivatization reaction was completed within 5 minutes without heating. The analytical features of the method are summarized in Table 2. The rapid one-step extraction–derivatization of the sample and the avoidance of the cleanup and evaporation steps significantly reduce the analysis time and solvent consumption. It is worth noting that the analytes [19,20] were derivatized without the need to use the drastic temperature and time conditions recommended in the literature (60 to 100°C for 0.5 to 3 hr).
Solid-phase extraction, elution, evaporation, and formation of TMS-deriva- tives is the most frequently used pretreatment method for the determination of cocaine and its metabolites by GC–MS. A CFS for the automated pretreatment
Drug Determinations in Biological Fluids 261
of urine samples for the determination of cocaine, ecgonine, ecgonine methyl ester, and benzoylecgonine was developed [21]. In this case, simultaneous elution and derivatization of the ecgonine and benzoylecgonine was impossible under the optimal conditions established, i.e., retention on RP-C18and elution with 2%
ammonium hydroxide (NH4OH) in isopropanol:CHCl3(25:75, v/v) spiked with the reagent bis(trimethylsilyl)acetamide (BSA). Therefore, both steps must be carried out separately (see Fig. 4) and the eluent removed by evaporation before the derivatizing reaction. Then, an on-line evaporation step must be implemented in the proposed manifold before the automated addition into the vial of BSA for redissolution and subsequent derivatization at 70°C for 15 minutes. The analytical features of the method are shown in Table 2.
Finally, a CFS for confirming the presence of benzodiazepines (through benzophenone formation) in previously screened positive samples was reported.
As hydrolysis of the urine samples was required in the screening method [41], hydrolyzed samples were also used for the GC–MS method [42]. This option resulted in several advantages, such as increased thermal stability of the benzo- phenones relative to the parent compounds, and a higher sensitivity, probably due to cleavage of the glucuronide bond, which allowed the use of sample volumes as low as 0.5 ml of urine. The hydrolysis was carried out in a discrete mode, as it required drastic conditions (100°C, 1 hr, 3 M HCl). The CFS developed for this purpose introduced two additional steps: on-line filtration of the urine samples through a home-made cotton column located at the inlet of the sample aspiration channel (see Fig. 4) and adjusting the pH of the analytes prior retention on an XAD-2 column, as sorption was favoured by an alkaline medium. Thus, the urine, in 3 M HCl, was merged with a 4 M NaOH stream to provide a pH greater than 12 after the mixing point prior to retention. Trichloromethane was used as eluent because of its increased selectivity. The analytical features of this method are summarized in Table 2.