Microsoft word - 047_lvi biological samples.doc

This is intended for Internal use ONLY and is not for Public Distribution Journal of Chromatography B. 729 (1999) 199-210 Evaluation of the programmed temperature vaporiser for
large-volume injection of biological samples in gas chromatography
M.W.J van Hout, R.A. de Zeeuw, J.P. Franke, G.J. de Jong*
Department of Analytical Chemistry and Toxicology, University Centre for Pharmacy, A. Deusinglaan 1, 9713 AV Groningen, Received 28 January 1999; received in revised form 1 April 1999; accepted 1 April 1999 Abstract
The use of a programmed temperature vaporiser (PTV) with a packed liner was evaluated for the injection of large volumes (upto 100 µl) of plasma extracts in a gas chromatograph. Solvent purity, which is essential when large volumes are injected into theGC system, was determined. Special attention was paid to the purity of the solvents used for the solid-phase extraction (SPE)procedure. For this SPE method, ethyl acetate was used as the extraction and reconstitution solvent, and thus the purity of theethyl acetate was critical, especially when a non-selective GC detector was applied. The liquid capacity and inertness ofdifferent packed liners were investigated. The liner packed with ATAS "A" (modified Chromosorb-based material with specialtreatment) was found to be the most suitable for the analysis of the tested drugs. Good linearity in response for variations involume and concentration was observed. A comparison was made between the applicability of flame ionisation detection(FID) and mass-selective detection (MSD). When 50-µl volumes of plasma extracts were injected with the PTV, the detectionlimits for secobarbital, lidocaine, phenobarbital and diazepam were about 50-times lower than when 1-µl volumes wereinjected. The detection limits of the tested compounds in plasma for injection of 50-100µl plasma extract are 5-10 ng/ml forGC-FID whereas plasma concentrations of 250 pg/ml can be detected using the selected ion monitoring (SIM) mode of aMSD. For non-selective GC-FID, the background from a 50-µl injection was substantially larger than with 1-µl injection as aresult of co-injected plasma matrix components and solvent impurities. These background effects were less with GC-MSD inthe total ion current mode and virtually absent with GC-MSD in the SIM mode. 1999 Elsevier Science B.V. All rights reserved. Keywords: Programmed temperature vaporiser; Diazepam; Lidocaine; Phenobarbital; Secobarbital 1. Introduction
consequently, drug concentrations in biological samples Increasing knowledge of the working mechanism of often are much lower than before. For the determination biologically active substances has led to the development of these lower levels in biological samples, analytical of potent drugs. Hence. lower dosages can be techniques with much higher sensitivity are needed. A administered to produce a therapeutic effect and, way to increase the sensitivity is to increase the amount of sample injected into the analytical system. In gas chromatography (GC) several techniques are Corresponding author. Tel.: +31-50-3633-337: fax. +31-50- 3637-582. available to perform large volume injections (LVIs) [1].
E-mail address: g.j.de.jong@farrn.rug.nl (GJ de Jong) On-column injection with the use of so-called retention 0378-4347/991$ -see front matter 1999 Elsevier Science BV. All rights reserved. PII: S0378-4347(99)00159-0 ATAS GL INTERNATIONAL B.V.
De Sleutel 9, 5652 AS, Eindhoven, The Netherlands Tel. +31 (0)40 254 95 31 Fax. +31 (0)40 254 97 79 E-mail: info@atasgl.com Internet: www.atasgl.com M.WJ. van Hout et al. / J. Chromatogr. B 7 2 9 (1 9 9 9 ) 199 - 21 0 gaps is currently the most common technique [1]. A second in water after in-vial liquid-liquid extraction and direct inj possibility for LVI is the loop-type interface [2], originally ection of the extracts with detection limits as low as 0.01 designed for the coupling of liquid chromatography (LC) µg/l, and polychlorinated biphenyls (PCBs) and polycyclic and GC. The main advantage of these techniques is that the aromatic hydrocarbons (PAHs) at the ppt-level [13]. complete sample is introduced into the GC column. Another application of the PTV is the residue analysis of However, this may also become a disadvantage since all 385 pesticides down to the 0.01 ppm concentration level in impurities are introduced into the GC system as well. A third option to allow LVI in GC is to use a programmed The purpose of the present work is to investigate the temperature vaporiser (PTV). Despite good results possibilities of the PTV coupled to GC for the analysis of obtained by Vogt and co-workers [3,4] in the late plasma extracts to provide lower detection limits for drugs.
seventies, only recently has PTV injection been applied as Special attention was paid to the impact of solvent a routine technique for environmental analysis [1]. impurities in view of the larger solvent volumes injected, to Besides conventional split/splitless injection, the PTV the liquid capacity and inertness of the PTV liners, and to can be used for several modes of LVI. The coupling of LC the degree of selectivity provided by flame ionisation and GC using the PTV was reviewed by Grob [5], and detection (FID) and mass-selective detection (MSD). recently interesting publications appeared on the same subject [6,7]. The PTV is often applied for this purpose because the packed liner generally has a larger liquid 2. Experimental
storage capacity than a retention gap. In addition, wettability is not very critical for the liquid retention and packing materials are more water-resistant than retention gaps with a silica backbone. The packing is more easily and rapidly heated than a retention gap [5]. Main reasons to couple LC with GC are that LC provides better Gas chromatographic analyses were performed with a resolution than more conventional techniques of sample Hewlett-Packard HP 5890 series II with AD or a GC-MSD preparation, and secondly, the possibility of automation system (HP 5971 series). A HP-5 30 m x 0.32 mm capillary through on-line coupling, which reduces or eliminates column with 0.25 µm film thickness was used for the manual sample preparation work and, therefore, reduces analyses with AD, whereas analyses with MSD were analysis time and improves accuracy and precision [5,7]. performed using a HP-5 MS 30 m x 0.25 mm column with The use of a PTV as the interface between LC and GC has 0.25 µm film thickness. The PTV injection system was an been demonstrated for the analysis of olive oil and for OPTIC 2 (ATAS International, Veldhoven, The environmental analysis [6,7]. The PTV is also used for Netherlands), which was equipped with 80 mm x 3.4 mm thermal desorption-pyrolysis of solid geochemical samples I.D. liners obtained from ATAS International. The liners (characterisation of oil and kerogens in source rocks) [8], were packed with either ATAS "A" packing (a modified and for on-line solid-phase extraction-thermal desorption Chromosorb-based material with special treatment, ATAS International), silanised glass wool (research grade, Serva, pesticides, chlorobenzenes and chlorophenols in aqueous Feinbiochemica, Heidelberg, Germany), or disposable capillaries for thin-layer chromatography (TLC) (nine Most applications of LVI are in the analysis of capillaries of 10 µl and two of 2 µl, cut at a length of 2 cm). environmental aqueous samples [1,9-13]. Pesticides were Plasma extractions were performed using Bond Elut determined in aqueous samples after SPE of samples of Certify cartridges (Varian, Harbor City, CA, USA), column 200 ml with concentrations between 0.2 and 5 ng/l by type LRC of 10 ml with 130 mg sorbent. A Visiprep system Steen et al. [ 12], whereas Teske et al. [ 13] determined (Supelco, Bellefonte, PA, USA) was used to apply vacuum triazines like atrazine, propazine, ametryne and simazine ATAS GL INTERNATIONAL B.V.
De Sleutel 9, 5652 AS, Eindhoven, The Netherlands Tel. +31 (0)40 254 95 31 Fax. +31 (0)40 254 97 79 E-mail: info@atasgl.com Internet: www.atasgl.com M.W.J. van Hout et al. / J. Chromalogr. B 7 2 9 (1 9 9 9 ) 199 - 21 0 The carrier gas for GC-FID and GC-MSD was helium. Acetonitrile and methanol (Lab Scan, Dublin, Ireland) The same temperature program was used for both methods.
were of HPLC quality. Acetone, hexane, acetic acid The starting temperature was 40°C, and after 3 min the glacial 100% (v/v), ammonia solution 25%, and KH2PO4 temperature was raised at 20°C/min to 215°C, followed by were all of analytical-reagent grade quality (Merck, an increase at 5°C/min to 230°C and a final increase at Darmstadt, Germany). Ethyl acetate (Reinst and 25°C/min to 290°C. This final temperature was maintained Suprasolv - for organic residue analysis) was obtained for 5-10 min. The detector temperature was 300°C. A from Merck (Darmstadt, Germany). Ethyl acetate Ultra column flow of 1.35 ml/min was used during analysis with resi-analysed (for organic residue analysis) was GC-FID and 0.48 ml/min with GC-MSD. The injector was purchased from Mallinckrodt Baker (Deventer, The set at 40°C and 10s after the evaporation of the solvent Netherlands). Water used during SPE was ultra pure (delay time) the temperature was raised with 5°C/s to 290°C. (Elgastat maxima, Salm en Kipp, Breukelen, The The end time was set at a time equal to the total run time of Netherlands). Secobarbital, phenobarbital (both BP one analysis. Other PTV settings are presented in Table 1. quality, Siegfried, Zofingen, Switzerland), lidocaine (Eur.
During analysis performed with GC-MSD in the total ion Ph., Holland Pharmaceutical Supply, Alphen A/D Rijn, current (TIC) mode an m/z range of 50-300 was monitored.
The Netherlands), and diazepam (Centrafarm, Etten-Leur, Using the selected ion monitoring (SIM) mode, the The Netherlands) were used as test compounds (Fig. 1) monitored m/z values were 86.0, 167.0, 204.0 and 256.0, and dissolved in ethyl acetate (for organic residue which corresponded to the most intense fragment of analysis, Mallinckrodt Baker). Stock solutions of 1 mg/ml lidocaine, secobarbital, phenobarbital and diazepam, were stored in the dark at 4°C. Stock solutions were mixed and then diluted with ethyl acetate (for organic SPE was performed as described previously [ 16] with residue analysis, Mallinckrodt Baker). The compounds of some minor modifications. The SPE column was activated the reference RI-mixture [15] were dissolved in ethyl with 2 ml methanol (2 ml/min), followed by conditioning of the SPE column with 2 ml of 0.1 M K2HPO4 buffer. pH 6(2 ml/min). Subsequently, 1 ml plasma, diluted with 4 mlK2HPO4 buffer, was brought on the column during approximately 1 min. Then the SPE column was washedwith 1 ml water and 0.5 ml of 1 M acetic acid (1.5 ml/min). The column was dried under vacuum for 4 min, Fig. 1. Structures of the test compounds. (A) secobarbital, ( B ) lidocaine. (C) phenobarbital, (D) diazepam. ATAS GL INTERNATIONAL B.V.
De Sleutel 9, 5652 AS, Eindhoven, The Netherlands Tel. +31 (0)40 254 95 31 Fax. +31 (0)40 254 97 79 E-mail: info@atasgl.com Internet: www.atasgl.com

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