3. BLOOD AND URINE ANALYSIS
3.1 Optimization of Solid-Phase Microextraction Sampling
Analysis of Inhalation Anesthetics in Urine
At equilibrium, analytes are not completely extracted from the matrix. Neverthe- less, reliable quantitative analysis is possible by SPME when sampling occurs under reproducible conditions. Optimization of SPME sampling is necessary when a high sensitivity is required, as in the case of halogenated anesthetics. In fact, using traditional static headspace sampling, only nitrous oxide, which is the main component of the anesthetic mixtures used in operating room theaters, can be determined. Urinary concentrations of anesthetics are in fact very low and a high sensitivity is required, especially for the determination of halogenates. Since inhalation anesthetics have never been studied before using SPME, we have opti- mized the kinetic of extraction and calculated the thermodynamic parameters (distribution coefficients and heats of adsorption), in order to characterize all the equilibria at the various interfaces of sample–headspace–fiber coating. We will use this example to show how to develop a SPME method for a completely new analyte. For the headspace sampling of anesthetics, a 75-àm Carboxen/PDMS- coated fiber was chosen for its very high affinity to halogenated compounds [34].
Samples (10 ml) were collected in 20-ml headspace vials containing 1 g NaCl and 200àl of H2SO49N. Analyses were performed by GC–MS using a capillary column with a divinylbenzene porous polymeric stationary phase (RT-QPLOT, 30 m⫻ 0.25 mm ID).
In principle, optimization of sampling conditions and method validation should be conducted in the real matrix. In the case of urine, sample collection is noninvasive and ethically acceptable. Large amounts of sample are usually available to perform an in-matrix method development.
3.1.1 Extraction Time Profile
Once the appropriate stationary phase and film thickness are chosen, taking into account the volatility and polarity of analytes, the development of a new SPME method starts with the determination of the time needed for the analytes to reach equilibrium between the matrix, the headspace (when present), and the fiber. The extraction time profile can be determined for each analyte by plotting the detector response versus the extraction time. When a further increase of the extraction time does not result in a significant increase in the response, the equilibration time is assumed to be reached. As an example, Figure 3 shows the extraction profile obtained for halogenated anesthetics and for the internal standard (IS) dichloromethane using a 75-àm-Carboxen/PDMS SPME fiber. The steady state is reached after 15 minutes for all the analytes and the IS. The compound chosen as IS should show a similar profile to those of the analytes. Sampling at equilib- rium reduces the possibility of errors and ensures reproducible data. Sampling at shorter but precisely measured extraction times is also possible and is recom-
236 Manini and Andreoli
Figure 3 Extraction time profiles of dichloromethane (I.S.), halothane, isoflurane and sevoflurane obtained using a 75-àm-Carboxen/PDMS SPME fiber. Sampling conditions:
25°C, no stirring.
mended when equilibration times are too long. High-molecular-weight com- pounds with high distribution coefficients, such as polycyclic aromatic hydrocar- bons (PAHs), require equilibration times of hours [12].
3.1.2 Effects of Temperature, Agitation, Salting, and pH
An increase in extraction temperature has the double effect of increasing the extraction rate but simultaneously decreasing the distribution constantKfs. In fact, at high temperatures the fiber starts releasing the adsorbed analytes faster than adsorbing. The temperature chosen should be a compromise between these two competitive effects. In general, sampling at room temperature is recommended for highly volatile compounds, for which the main effect is theKfsdecrease, while for low volatiles the increase of the extraction rate is the most relevant effect, which is positively influenced by higher temperatures. A modified SPME system, which allows the sampling with an internally cooled fiber from a heated system, has been proposed to enhance the sensitivity [35], but it is not commercially available. Heating the sample can also be useful to help the release of the analytes from a solid matrix, if both the analytes and the matrix are stable at higher temper- atures.
Static sampling is simpler but is limited to volatile analytes and headspace SPME. Agitation of the sample can help the mass transport between the bulk and the fiber, leading to a response increase for low volatile compounds, such as PAHs [28]. Agitation can be obtained by magnetic stirring, intrusive stirring,
Solid-Phase Microextraction 237
vortex, fiber movement, flow through, or sonication [10]. Stirring might have the drawback of heating up of the sample. Anesthetics are very volatile compounds, permanent gases or liquids with very low boiling temperatures; for these reasons, we sampled at room temperature without agitation.
Salting can increase the extraction efficiency, especially in the case of polar compounds [14]. Concentrations of salt above 1% are reported to cause a substan- tial increase in extraction efficiency. Saturation with salt can be used to normalize random salt concentrations in natural matrices [26]. We found that a 10% of salt (w/v) added to urine samples led to a 26 to 35% increase in sensitivity for inhala- tion anesthetics, whereas a further addition of salt (up to 50%) did not further lower the detection limit. Besides salts, other additives such as nonvolatile acids [35] or water [36] can be used to enhance the extraction of analytes from very complex matrices.
pH control is recommended when dissociable species, acids or bases, are analyzed by SPME [8]. Since only neutral species are extracted, the pH should be two units below or above the pK for acid or basic analytes, respectively. Once the pH of the sample has been adjusted, only headspace sampling is possible [26].
3.1.3 Desorption Temperature and Time
Besides the sampling parameters, the desorption temperature and desorption time also need optimization to avoid sample carryover on the fiber. The desorption temperature must be at least as high as the highest boiling point of the compounds in the mixture. Since desorption becomes faster when the desorption temperature increases, a rapid way to optimize desorption conditions is to set the injector temperature at the maximum allowable temperature (determined by the coating) and then to adjust the desorption time to obtain quantitative desorption in a single run. Carryover can be determined by injecting the fiber again, immediately after the end of the chromatographic run. Compounds with very high molecular weight may lead to carryover problems. Thermal desorption of anesthetics was per- formed at 240°C for 16 minutes.
3.1.4 Partition Coefficients and Heat of Adsorption
The efficiency of the extraction process and the sensitivity of SPME depend on the fiber coating–sample distribution constant,Kfs, a thermodynamic parameter which expresses the affinity of the fiber coating for a target analyte.Kfsvalues for organic compounds may vary widely (3 to 4 orders of magnitude) depending on the coating and the thickness of the SPME fiber. Table 2 compares literature Kvalues obtained with different coatings for BTEX, the most investigated class of organic compounds. For air samples, coating–gas distribution constants,Kfg
can be estimated using GC retention times on a column with stationary phase
238 Manini and Andreoli
Table 2 Comparison of Distribution ConstantKValues Obtained with Different Fiber Coatings andKowfor BTEX
K K
PDMS [12] Bonded phases [6] K
Carboxen [34] Kow
100àm 7àm C-8 70àm C-18 70àm 80àm [34]
benzene 199 97 31.8 10.4 8550 135
toluene 758 383 96.3 42.3 10270 537
ethylbenzene 2137 519 n.d. n.d. 8300 1412
m-xylene 2041a 448a 227.9 201.7 8600 1412
o-xylene 1819 365 105.6 137.2 9040 1318
am⫹p-xylene.
identical to the fiber coating or using the linear temperature-programmed reten- tion index system [37] or Kovats retention indices [38]. In the case of aqueous liquid samples, the coating–water distribution constants,Kfw, estimated for direct SPME extraction have been correlated with the octanol–water,Kowpartition coef- ficients [39]. The coefficients calculated in pure water or air are modified in the presence of the sample matrix [40].
In headspace mode,Kfsdetermines the absolute SPME response and can be calculated as:
Kfs⫽Kf hKhs⫽KfgKgs (5)
where Khs is directly related to the Henry’s constant of the analyte [1].Kf hhas been calledK, the calibration factor [41].
In principle, it is not necessary to calculate the partition coefficients, when quantitative analysis is performed with the standard addition method or with iso- topically labeled standards. On the other hand, calibration of the sampling device is not necessary when the analyte’sKat various temperatures is known [37,42].
In any case, it is always advisable to determine partition coefficients, since knowl- edge of them may help to better understand all the phenomena occurring at the various interfaces and to predict the effects of a modification of the system.
In the case of halogenated anesthetics, we obtained very highKfsvalues (110,000 to 190,000). Therefore, we could reach the required sensitivity for our biological monitoring purposes. As expected, both the MS response factor and the affinity of Carboxen/PDMS for nitrous oxide were lower, leading to a 1000- fold lowerKfsvalue (103). Figure 4 shows an SPME–GC–MS chromatogram of a urine sample from an occupationally exposed subject. Despite the low urinary concentrations, the peaks of halothane (1.1àg/L) and isoflurane (1.3àg/L) are
Solid-Phase Microextraction 239
Figure 4 The SPME–GC–MS analysis of a urine sample from a worker exposed to anesthetics. Peak identification: 1, nitrous oxide; 2, I.S.; 3, isoflurane; and 4, halothane.
Figure 5 Plot of lnKversus 1000/Tfor isoflurane and halothane.
240 Manini and Andreoli
clearly detectable, whereas in the case of static headspace sampling, only the peak of nitrous oxide (5.5àg/l) was present.
The heat of absorption,∆H, can be determined by measuringKfsat different temperatures and by plotting the lnK versus the inverse absolute temperature.
The slope of the straight line is⫺∆H. Figure 5 shows the experimental plots obtained in the case of isoflurane and halothane, the calculated heats of adsorption being⫺20.0 and⫺24.2 kJ/mol, respectively. A negative∆H, typical of volatile compounds, reflects the exothermic nature of the adsorption process: efficient sampling occurs at lower temperatures.