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Supported liquid membrane extraction coupled in-line to commercial capillary electrophoresis for rapid determination of formate in undiluted blood samples
Accepted Manuscript Title: Supported liquid membrane extraction coupled in-line to commercial capillary electrophoresis for rapid determination of formate in undiluted blood samples Author: Pavla Pant˚ucˇ kov´a Pavel Kub´anˇ Petr Boˇcek PII: DOI: Reference:
S0021-9673(13)00838-8 http://dx.doi.org/doi:10.1016/j.chroma.2013.05.058 CHROMA 354375
To appear in:
Journal of Chromatography A
Received date: Revised date: Accepted date:
2-4-2013 23-5-2013 24-5-2013
Please cite this article as: P. Pant˚ucˇ kov´a, P. Kub´anˇ , P. Boˇcek, Supported liquid membrane extraction coupled in-line to commercial capillary electrophoresis for rapid determination of formate in undiluted blood samples, Journal of Chromatography A (2013), http://dx.doi.org/10.1016/j.chroma.2013.05.058 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Supported liquid membrane extraction coupled in-line to commercial capillary
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electrophoresis for rapid determination of formate in undiluted blood samples
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Pavla Pantůčková, Pavel Kubáň*, Petr Boček
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Institute of Analytical Chemistry of the Academy of Sciences of the Czech Republic, v. v. i.,
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Veveří 97, CZ-60200 Brno, Czech Republic
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Dr. Pavel Kubáň, e-mail: [email protected], Tel: +420 532290140, Fax: +420 541212113, Institute
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of Analytical Chemistry of the Academy of Sciences of the Czech Republic, v. v. i., Veveří 97,
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CZ-60200 Brno, Czech Republic
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Keywords: blood samples; capillary electrophoresis; formate; in-line sample pretreatment;
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methanol intoxication; supported liquid membranes
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Abstract
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A cheap, disposable sample pretreatment device with planar supported liquid membrane (SLM)
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was proposed, assembled and placed into an autosampler carousel of a commercial capillary
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electrophoresis (CE) instrument for automated pretreatment and analysis of formate in undiluted
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whole blood and serum samples. All analytical procedures except for filling the pretreatment
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device with donor and acceptor solutions, i.e. extraction across SLM, injection of the extracted
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sample and CE-UV determination of formate, were performed fully automatically. The
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pretreatment device required only µL volumes of blood sample and organic solvent per
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extraction and was disposed off after each extraction. Good repeatability of peak areas ( 7.7%)
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and migration times ( 1.5%), linear relationship (r2 = 0.998 – 0.999) and limits of detection (
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35 µM) were achieved. The overall analytical process including blood withdrawal, filling the
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SLM device with respective solutions, extraction of blood sample, injection into separation
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capillary and CE separation of formate from other anions took less than 4 min. The method was
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proved useful by direct determination of elevated formate concentrations in undiluted serum
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samples of a methanol intoxicated patient. Due to its compatibility with currently commercially
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available CE instrumentation, disposability of extraction devices, minimum sample
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handling/consumption, and short extraction/analysis times, the developed method might be
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attractive for rapid diagnosis of methanol poisoning in clinical and toxicological laboratories.
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1. Introduction
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Clinical samples, such as human serum and whole blood, contain usually limited amount of
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target analytes and high concentrations of bulk components, which often deteriorate performance
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of capillary electrophoresis (CE). Most serious complications arise from the presence of major
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blood components, e.g. proteins, lipids and salts and additionally of red blood cells in the case of
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whole blood. Proteins, lipids and particulate matter tend to adhere to the inner surface of
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separation capillaries and have severe effect on CE performance, selectivity and sensitivity [1-4].
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Blood samples are therefore only scarcely injected without sample pretreatment and in most CE
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applications they are pretreated in order to eliminate their matrices [5-9]. The pretreatment is
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normally performed using stand-alone sample pretreatment instrumentation and the cleaned-up
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and, in most cases, also preconcentrated samples are then off-line transferred to CE for injection.
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However, the off-line transfer may be inconvenient due to the risk of sample contamination
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induced by manual handling, loss of analytes and need for unattended operation. Set-ups
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allowing for direct coupling of pretreatment and analytical techniques are therefore beneficial
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and reports on direct coupling of various pretreatment devices to CE were comprehensively
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reviewed by Valcárcel et al. [5,6]. Vast majority of these set-ups was realized with lab-made CE,
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which enabled the required flexibility for coupling the pretreatment techniques to CE.
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Contrary to lab-made CE, direct coupling of sample pretreatment techniques to commercial CE
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necessitates additional alterations of the pretreatment devices in order to enable their
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compatibility with CE injection systems and/or sample vials/carousels [7]. Direct coupling of
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sample pretreatment to CE using external instrumentation, such as flow-through programmable
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arm [10-12] and pump system [13,14], was reported. However, in order to transfer the pretreated
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samples to CE, the external instrumentation must be sophisticated and must be precisely
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synchronized with the movement of the autosampler carousel. The need for the external
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instrumentation and its synchronization with CE can be overcome by using sample pretreatment
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in-capillary [15-17], in-vial [18-21] or at-capillary tip [22,23]. These approaches are particularly
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interesting since the entire pretreatment, injection and separation process is performed in an
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automated manner inside the commercial CE. Nevertheless, in-capillary sample pretreatment
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suffers from the fact that complex samples have to be flushed through the separation capillary
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during sample loading. To avoid direct contact of their matrix components with the capillary
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inner walls, samples were usually loaded off-line [15-17]. In-vial arrangement eliminates the
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contact of the separation capillary with the sample matrix by inserting a supported liquid
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membrane (SLM), a thin porous polymeric membrane impregnated with water immiscible
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solvent [24-27], between the aqueous donor and acceptor solution. However, fabrication of
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SLM-based pretreatment devices, which are compatible with commercial CE instruments, is
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elaborate and does not allow their disposable use. Regular conditioning of the SLM is therefore
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necessary and limited life-time of these devices was reported for pretreatment of complex
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samples [18-20]. The major drawback of the set-up where a small aqueous acceptor droplet is
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formed at the separation capillary tip and covered by a thin organic layer is limited stability of
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the organic layer in aqueous solutions [22,23]. Moreover, stability of the entire droplet is reduced
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when the donor solution is agitated, which is necessary to ensure constant replenishment of
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analytes at the donor/acceptor interface.
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In this manuscript, a sample pretreatment device with SLM [21] was in-line coupled to
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commercial CE for direct determination of formate in blood samples. Formate is the major
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metabolite of methanol dehydrogenation in human bodies, can be accumulated therein and is
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primarily responsible for the toxicity in methanol poisonings [28]. Analytical techniques, which
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are currently used for determination of formate in suspected methanol poisonings, are headspace
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gas chromatography (GC) with flame ionization or mass spectrometric detection [29-31] and
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enzymatic methods combined with spectrophotometry [32,33]. However, GC methods are time-
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consuming and require sample derivatization, and enzymatic methods necessitate considerable
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incubation times and reagents are costly. Two CE methods were reported recently for
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determination of formate in human blood samples associated with methanol poisonings, which
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offer reduced analysis times and costs [34,35]. Note however, that lab-made CE instrumentation,
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which must be manually operated by trained personnel and is not acceptable for routine use in
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clinical laboratories, was used in these reports and formate was detected with conductivity
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detectors, which are not standard equipment in commercial CE systems. Determination of
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formate was performed in blood serum [34], which additionally necessitates whole blood
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clotting, centrifugation and serum collection prior to analysis and therefore considerably
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increases the total analysis time. Microdialysis membranes and units, which are rather expensive
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and cannot be used as disposable extraction devices, were used for sample pretreatment in [35].
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Moreover, additional sample handling, i.e. 100-fold [34] and 10-fold [35] dilution, was required
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in order to minimize the effect of sample matrix and to reduce sample consumption.
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In this report, a new method was developed for direct analysis of formate in µL volumes of
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undiluted blood samples based on in-line coupling of disposable sample pretreatment device to
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commercial CE equipped with standard UV-Vis detector. The overall analysis time, including
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whole blood withdrawal and automated sample extraction, injection, CE separation and
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quantification of formate, was less than 4 min. Beckman P/ACE 5000 series as well as Agilent
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7100 CE instruments were proved useful for direct analysis of formate in raw blood samples.
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2. Materials and methods
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2.1. Instrumentation
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2.1.1. Sample pretreatment devices
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A sample pretreatment device, which is compatible with injection system of Beckman P/ACE
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5000 series CE instruments (Beckman Instruments, Fullerton, CA, USA), and its application in
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pretreatment of complex samples was described in detail previously [21]. Volume of acceptor
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and donor solution was 15 L, volume of organic solvent for SLM impregnation was 10 L and
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complete pretreatment device was disposed off after each extraction in the actual measurements.
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Optimization of the analytical method for direct coupling of SLM extractions to commercial CE
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for formate analyses was performed with the Beckman P/ACE CE instrument.
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The currently most popular commercially available CE system (7100 CE, Agilent Technologies,
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Waldbronn, Germany) was also used for direct coupling to SLM extractions. Volume and size of
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sample vials are reduced in Agilent 7100 CE compared to Beckman P/ACE 5000 instruments
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and the SLM device had to be down-scaled accordingly (see Fig. 1). Donor and acceptor units
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were pre-cut from 200 µL polypropylene (PP) micropipette tips (Kartell Spa, Noviglio, Italy) to
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fit the internal diameter of the Agilent sample vials (Part No. 5182-0567). PP membranes
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(Accurel PP 1E R/P, Membrana, Wuppertal, Germany, 100 µm thick, average pore size 0.1 µm)
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were pre-cut from the Accurel PP sheet using a 11 mm cork borer. SLM devices were assembled
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according to the following procedure, which was performed routinely in ca. 1 min. First, the PP
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membrane was impregnated with 10 L of an organic solvent. Second, the membrane was placed
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on top of the donor unit and firmly pressed against the bottom of the acceptor unit. Third, the
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donor and acceptor unit was filled with 10 µL of donor and acceptor solution, respectively, and
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measurement of extraction time was started. Note that the units are filled only partially and
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empty space was left below and above the donor and acceptor solution, respectively (Fig. 1), for
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ease of manipulation. Fourth, the SLM device was accommodated in the sample vial, which was
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placed into CE autosampler. The issue of prefabrication and long-term storage of SLM devices
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with impregnated membranes was not examined as it is beyond the scientific scope of this
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manuscript.
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In addition to reduced size of sample vials, geometry of the HV electrode and its position relative
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to the injection end of the separation capillary is different in Agilent CE instruments. The
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capillary is placed in centre of a tubular HV electrode and the capillary tip and electrode rim are
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positioned close to the vial bottom during injection (see Fig. 1B). For this reason, a 5 3 mm
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cylinder was cut from soft polyurethane (PU) foam (unknown producer) and placed at the sample
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vial bottom below the pretreatment device. During sample injection, the sample vial is lifted up
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and the injection end of the separation capillary touches the SLM. As the SLM device is
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positioned on the soft PU foam, the device can be easily pressed down into the foam (see Fig.
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1B), which releases the pressure induced during the upward movement of the sample vial and
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avoids perforation of the SLM. Moreover, it simultaneously ensures that the injection end
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remains in direct contact with the SLM during injection. The injection end of the separation
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capillary overlaps the rim of the tubular HV electrode by ca. 0.5 mm (see Fig. 1). This position
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differs from that previously optimized and used for Beckman P/ACE instruments. The reason is
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better repeatability of injections compared to those observed when the capillary tip and the
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electrode rim were levelled off or when the electrode rim overlapped the capillary tip.
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After extraction, the pretreatment device was removed from the sample vial and was disposed
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off. A new set of donor/acceptor units and a new SLM was used for each extraction. The analyte
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transfer across the SLM, expressed as recovery, was calculated as described previously [36].
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2.1.2. Capillary electrophoresis
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Automated CE instruments P/ACE 5000 (Beckman Instruments) and 7100 CE (Agilent
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Technologies) equipped with UV-Vis detector (operated at 214 nm in indirect mode) were used.
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Indirect absorbance signals were software-wise inverted in order to obtain positive peaks.
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Electrophoretic separations using P/ACE instrument were performed in a fused-silica (FS)
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capillary (75 µm ID/365 µm OD, 27/20 cm total/effective length, Polymicro Technologies,
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Phoenix, AZ, USA). Prior to the first use, the bare capillary was treated as described recently
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[21]. Between two CE runs, the capillary was rinsed with DI water and BGE solution at 20 psi
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(1379 mbar) for 18 s each. Anionic analytes were analyzed in anionic mode without reversal of
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EOF, i.e. with cathode at the injection side. CE separations were performed at a constant voltage
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of – 30 kV. Capillary temperature was maintained at 25°C and injections were performed
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hydrodynamically at 0.5 psi (34.5 mbar) for 5 s. The CE instrument was controlled and analytical
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signals were processed using Beckman P/ACE Station Version 1.0 software.
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The Agilent 7100 CE system was operated at a potential of – 30 kV applied at the injection side
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of the separation capillary for all runs. Separation capillary used was a FS capillary (75 µm
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ID/375 µm OD, 31/22.5 cm total/effective length, Polymicro Technologies). Conditioning of the
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bare capillary and rinsing procedure between two CE runs were performed according to the
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procedure described above at a pressure of 950 mbar. Capillary temperature was maintained at
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25°C and injections were performed hydrodynamically at 50 mbar for 5 s. The CE instrument
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was controlled and analytical signals were acquired by ChemStation CE software.
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2.2. Reagents, BGE solutions, standards and blood samples
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All chemicals (reagent grade) were purchased from Pliva-Lachema, Brno, Czech Republic;
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Sigma, Steinheim, Germany; and Fluka, Buchs, Switzerland. DI water with resistivity higher
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than 18 Mcm was prepared by exchange of ions in a mixed-bed ion exchanger water
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purification system G 7749 (Miele, Gütersloh, Germany). 1 M stock solution of Cl- was prepared
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from NaCl. Stock solutions of other inorganic anions (100 mM) were prepared from their Na+ or
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K+ salts. Stock solutions of organic anions (100 mM) were prepared from their Na+ or K+ salts or
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from organic acids. All stock solutions were kept refrigerated at 4C. Standard sample solutions
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of the inorganic and organic anions were freshly prepared from these stock solutions and were
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diluted with DI water. Human serum albumin (HSA) was purchased from Sigma. BGE solutions
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for CE measurements were prepared from DL-mandelic acid (Sigma) and -aminocaproic acid
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(Pliva-Lachema) and were kept refrigerated at 4C. The optimum separation of formate from
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other inorganic/organic anions was achieved in BGE solution consisting of 15 mM DL-mandelic
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acid and 25 mM -aminocaproic acid (pH 4.35). SLMs were prepared by impregnating PP
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membranes (Membrana) with methanol, 1-butanol and 1-pentanol (Fluka). Human serum and
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whole blood samples were collected from healthy volunteers at the Institute of Analytical
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Chemistry; a written informed consent was signed by the volunteers before the experiments.
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Serum samples were prepared from clotted intravenous blood by centrifugation at 6000 rpm for
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10 min. Whole blood samples were collected either from a finger-stick (for instant analysis) or as
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anti-coagulated (EDTA addition) intravenous blood. Serum samples of a patient diagnosed and
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treated with acute methanol poisoning were donated by Prof. Robert Bocek from Department of
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Anaesthesiology, Resuscitation and Intensive Care, Hospital and Policlinics Havířov, Czech
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Republic. All blood samples were used undiluted, stored at –20°C and allowed to warm up to the
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ambient temperature prior to analysis.
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3. Results and discussion
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3.1. BGE solution
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Basic configuration of most commercially available CE instruments consists of a CE module and
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a UV-Vis detector. The selection of BGE solutions for analysis of formate in blood samples was
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therefore optimized with respect to detection in UV-Vis region. Formate posses no chromophore
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and is therefore detected in indirect mode using BGE solutions composed of highly UV-
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absorbing species. A BGE solution consisting of DL-mandelic and -aminocaproic acid was
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shown suitable for CE-indirect UV determination of various inorganic and organic anions
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previously [37] and was adopted for our measurements. CE separations of formate from other
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anions, which might be present in blood samples and might interfere with formate determination,
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i.e., chloride, sulfate, nitrite, nitrate, oxalate, tartrate, fumarate, citrate, maleate, malate,
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malonate, lactate, carbonate, phosphate and acetate, were predicted in various BGE solutions by
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using a modelling freeware Peakmaster [38]. BGE solutions were theoretically examined in the
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range of 5-15 mM for DL-mandelic acid and of 6-60 mM for -aminocaproic acid (pH 4 – 4.9).
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CE separations in selected BGE solutions were subsequently verified experimentally. Due to the
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low pH values of the examined BGE solutions, reversal of EOF was not required and fast
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separations of most analytes were achieved in counter-electroosmotic separation mode.
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Separation of a standard solution of inorganic and organic anions in the optimum BGE solution
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(15 mM DL-mandelic acid and 25 mM -aminocaproic acid at pH 4.35) is shown in Fig. 2.
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Formate is baseline separated from the fast inorganic anions (chloride, sulfate, nitrite and
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nitrate), and from oxalate, tartrate and fumarate. All other anions from the above mentioned list
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exhibited even slower migration than fumarate, did not interfere with formate determination, and
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were detected at times longer than 1.8 min.
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3.2. Donor and acceptor solution
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Donor solutions were standard solutions of respective analytes and NaCl, blood serum and whole
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blood. The actual composition of standard solutions is mentioned in the relevant text further on.
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In all cases, donor solutions were used undiluted and untreated. This minimizes the possibility
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for sample contamination, which might be implied by sample dilution and/or addition of
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reagents, and ensures very fast sample handling and sufficient sensitivity of the analytical
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method. Acceptor solutions were pure DI water. It has been described previously that for
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extractions performed directly at the SLM surface, pH gradients across the SLM offer no
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advantage over the simplest possible set-up with pure DI water as acceptor solution [21,36].
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3.3. Organic solvent used in SLM
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Extractions of small inorganic and organic anions across SLMs formed by impregnation of PP
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membranes with short-chain aliphatic alcohols were reported previously [39-41]. Methanol, 1-
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butanol and 1-pentanol were therefore examined as organic solvents in SLMs. Recoveries after
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60 s extraction of 1 mM formate in a standard solution of 100 mM NaCl were approximately
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18%, 4% and 6% for methanol, 1-butanol and 1-pentanol, respectively. In addition to the SLMs,
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dry PP membrane and a 500 Da molecular weight cut-off dialysis membrane [35] were
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examined. Formate was not transferred across the dry PP membrane as the hydrophobic
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membrane completely separated the donor and acceptor solution and ion transfer between the
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two solutions was not feasible. Transfer of formate across the dialysis membrane was
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comparable to that across the SLM impregnated with methanol, however, at the expense of
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increased chloride transfer and ca. 1000-fold higher costs of the membrane.
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Repeatability of the formate transfer was therefore further examined across the SLMs only. Most
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repeatable results were observed for methanol, which is consistent with the previous findings
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[40]. The alcohols are evenly distributed in the pores and on the surface of the PP membrane
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during the SLM impregnation. Nevertheless, as the exceeding organic solvent partially
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evaporates from the membrane surface during manipulation with the SLM, the thickness and
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uniformity of the liquid membrane, which are responsible for the analyte transfer across SLMs,
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vary for individual solvents. Consequently, this has a direct bearing on the extraction
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performance and influences the total transfer (expressed as recoveries) and transfer repeatability.
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Best extraction performance achieved for SLMs impregnated with methanol may be explained
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by the fact that methanol evaporates most rapidly from the SLM surface and remains only in the
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membrane pores, which ensures rather constant SLM properties. Methanol was therefore applied
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as the liquid membrane in all subsequent SLM extractions.
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3.4. Extraction time
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It has been shown recently that minute extraction times can be achieved for stagnant diffusion
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when the diffusive layer is minimized and pretreated samples are injected directly from the SLM
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surface [36]. For lab-made CE systems the extraction and injection process can be synchronized
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and instant CE injection can be performed almost at the same time when the pretreatment device
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is assembled [41]. This is, however, not possible in commercial CE instruments and certain time
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period is required to perform CE initialization, flushing and injection after assembling the SLM
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device. The minimum extraction time was set at 60 s and time program of the CE system was
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adjusted in order to perform the first injection 60 s after the SLM device was filled with donor
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and acceptor solutions. The SLM device was then retained in the autosampler carousel and
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subsequent injections were performed from the device at 150 s intervals. Fig. 3 depicts analysis
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of a standard solution of 1 mM formate and 100 mM NaCl extracted for 60, 210 and 360 s.
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Consistent results were achieved for peak areas of formate injected at extraction times up to 10
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min and gradual decrease was observed for subsequent injections at extraction times of 20 and
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30 min. The lower analytical signals are likely to be caused by gradual dissolution of the liquid
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membrane, which may result in considerable changes and dilution of the aqueous solution and
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analyte concentration at the injection (acceptor) side of the SLM. Maximum recovery was
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obtained already for the first measurement (60 s extraction time) and no further increase of the
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formate recovery was observed for subsequent measurements. These experiments demonstrate
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that the extraction process at the SLM surface has reached equilibrium within the first 60 s.
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Extraction time of 60 s was used for all subsequent extractions.
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3.5. Interferences
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Composition of human blood is rather consistent and variations in concentrations of most blood
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constituents are less than 10% in healthy individuals [42]. Human blood consists of major (e.g.
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HSA, Na+ and Cl-) as well as minor components and effect of the major ones on the extraction
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performance was investigated. Standard solutions of 1 mM formate were prepared in DI water
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containing HSA (70 g/L) and various concentrations of NaCl (10 – 100 mM). Performance of the
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CE separation was not affected by the presence of HSA in donor solutions, which confirms the
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generally known fact that liquid membranes can efficiently eliminate large molecules, such as
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proteins. Addition of increasing concentrations of NaCl showed measurable changes in formate
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recoveries, which gradually decreased from 35% (10 mM NaCl) to 18% (100 mM NaCl). This is,
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however, not surprising since the transfer of analyte ions across the methanol-based SLMs
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depends on concentration ratios of analyte/matrix ions [40]. A gradual increase of chloride peak
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was also observed during these experiments, demonstrating higher transfer of NaCl due to its
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elevated initial concentrations. Recovery values for extractions of standard solutions consisting
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of 90 and 110 mM NaCl (typical range for chloride in blood) and for extractions of undiluted
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serum and whole blood samples, all spiked with 1 mM formate, ranged between 17 and 21%.
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Obviously, the variations of the NaCl concentrations in real blood samples do not interfere with
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sufficient repeatability of the formate recoveries. Hence, the analytical method is well applicable
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to extractions and analyses of formate in blood samples.
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3.6. Analysis of human blood samples and analytical parameters of the method
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Electropherograms of blank samples (100 mM NaCl, human serum and human whole blood from
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a finger-stick) measured after extraction for 60 s are depicted in Fig. 4A. Analytical signals of
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formate were below the limit of detection of the method and fall within the reference range (0 –
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400 µM) reported in healthy individuals [43,44]. The blood samples were subsequently spiked
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with 1, 10 and 20 mM of formate and gradual increase of the formate peak was observed as is
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depicted for human serum in Fig. 4B.
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Table 1 summarizes analytical parameters of the presented SLM-CE method. Standard solution
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of 100 mM NaCl, blood serum and whole blood, all spiked with 1 mM formate, were used for
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repeatability measurements. RSD values of peak areas of 5 independent extractions were within
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the range 5.5 – 7.7% and average recoveries ranged between 18% and 21%. Inter-day and intra-
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day repeatability of peak areas was below 7.7% and 11.8%, respectively. Calibration curves were
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plotted for the standard solution consisting of 100 mM NaCl and blood samples, which were all
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spiked with formate concentrations in 0.5 – 20 mM range. Calibrations were linear in the whole
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concentration range with correlation coefficients between 0.998 and 0.999. Limits of detection
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(LODs), defined as 3 S/N ratio, were between 30 – 35 µM for the three different matrices. Note,
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that even though the method recoveries are relatively low, its sensitivity is sufficient since the
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LODs are about 2-3 orders of magnitude below the formate concentrations associated with
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methanol intoxications.
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3.7. Analysis of human serum of a methanol intoxicated patient
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Serum samples of a patient diagnosed with acute methanol poisoning, which were withdrawn
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after 3 and 4 h of hemodialytic treatment, were analyzed. Concentrations of formate determined
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in these samples were 12.3 ± 0.5 mM and 10.4 ± 0.4 mM, respectively, and correlate well with
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the previous results on determination of formate in the same serum samples using CE-C4D [34]
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and microelectrodialysis-CE-C4D [35]. The elevated formate concentrations in the early stage of
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the treatment demonstrate serious methanol intoxication of the patient. The subsequent decrease
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(between 3 and 4 h of treatment) follows the formate half-life in serum under hemodialytic
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treatment [45]. Fig. 5 depicts analysis of the patient’s serum sample after 4 h of hemodialytic
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treatment using the P/ACE 5000 series CE (trace a) and Agilent 7100 CE (trace b). Apparently,
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there are only insignificant differences between the two records and the method can easily, with
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just slight alterations to the SLM device design, be implemented into various commercial CE
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instruments.
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329 330
4. Conclusions
15 Page 15 of 29
Disposable sample pretreatment devices compatible with injection systems of commercial CE
332
instruments were proposed, assembled and applied to rapid CE-UV analyses of formate in
333
human whole blood and serum. SLM was sandwiched between two conical segments of the
334
pretreatment device and formed a selective barrier between donor and acceptor solutions.
335
Equilibrium between the formate concentrations at the SLM surface was established rapidly and
336
extracted formate was injected into CE separation capillary directly from the acceptor side of the
337
SLM. Stable CE performance was achieved since the SLM eliminated high molecular mass
338
matrix components and enabled only transfer of small molecules. The developed method may
339
find wide application in clinical and toxicological laboratories for the following reasons. The
340
complete extraction process, injection of extracted sample and CE-UV analysis of extracted
341
analytes
342
instrumentation. The method is applied to analysis of few µL of undiluted whole blood and no
343
additional sample handling, such as blood clotting, centrifugation, serum collection and dilution,
344
is necessary. Cost of the pretreatment device is negligible (less then 0.02 € per device) and the
345
entire pretreatment device is disposed off after sample extraction. Total analysis time including
346
whole blood withdrawal, sample extraction, and CE injection/separation/quantification of
347
formate is less than 4 min. Characteristics of the developed method offer an unrivalled analytical
348
performance for formate analyses as can be seen in Table 2.
cr
us
automatically
an
fully
using
M
performed
commercially
available
analytical
te
d
is
Ac ce p
349
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331
350
Acknowledgments
351
Financial support from the Academy of Sciences of the Czech Republic (Institute Research
352
Funding RVO:68081715) and the Grant Agency of the Czech Republic (Grant No. 13-05762S) is
353
gratefully acknowledged. Prof. Robert Bocek (Department of Anaesthesiology, Resuscitation
16 Page 16 of 29
354
and Intensive Care, Hospital and Policlinics Havířov, Czech Republic) and Dr. Petr Kubáň
355
(CEITEC, Masaryk University, Brno, Czech Republic) are also acknowledged for donation of
356
serum samples of a patient treated with acute methanol poisoning.
Ac ce p
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17 Page 17 of 29
357
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J. Viinamäki, I. Rasanen, E. Vuori, I. Ojanperä, Forensic Sci. Int. 208 (2011) 42.
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K.E. Hovda, P. Urdal, D. Jacobsen, J. Anal. Toxicol. 29 (2005) 586.
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L. Křivánková, P. Pantůčková, P. Gebauer, P. Boček, J. Caslavska, W. Thormann,
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Electrophoresis 24 (2003) 505.
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[38]
B. Gaš, M. Jaroš, V. Hruška, I. Zusková, M. Štědrý, LC-GC Europe 18 (2005) 282.
404
[39]
I.K. Kiplagat, K.O.D. Thi, P. Kubáň, P. Boček, Electrophoresis 32 (2011) 3008.
405
[40]
T.Y. Tan, C. Basheer, K.P. Ng, H.K. Lee, Anal. Chim. Acta 739 (2012) 31.
406
[41]
P. Kubáň, I.K. Kiplagat, P. Boček, Electrophoresis 33 (2012) 2695.
407
[42]
L.A. Kaplan, A.J. Pesce, S.C. Kazmierczak, Clinical Chemistry: Theory, Analysis,
ip t
403
cr
Correlation, Mosby Inc., St. Louis, Missouri, 2003.
408
[43]
R.P. Hanzlik, S.C. Fowler, J.T. Eells, Drug Metab. Dispos. 33 (2005) 282.
410
[44]
K.E. Hovda, K.S. Andersson, P. Urdal, D. Jacobsen, Clin. Toxicol. 43 (2005) 221.
411
[45]
K.E. Hovda, D. Jacobsen, Human Exp. Toxicol. 27 (2008) 539.
an
us
409
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412
20 Page 20 of 29
412
Figure captions
413
Fig. 1. SLM pretreatment device coupled directly to Agilent 7100 CE instrument. (A) sample
414
vial before sample injection, (B) sample vial during injection of the pretreated sample.
ip t
415
Fig. 2. Electropherogram of a standard solution of anionic analytes. CE measurements were
417
performed with the P/ACE 5000 CE instrument. CE conditions: BGE solution; 15 mM DL-
418
mandelic acid and 25 mM -aminocaproic acid at pH 4.35, voltage, –30 kV; injection, 0.5 psi for
419
5 s. Standard solution contained 0.2 mM chloride, 0.05 mM sulfate, 0.5 mM oxalate, 0.1 mM
420
nitrite, nitrate, formate, fumarate and tartrate.
an
us
cr
416
421
Fig. 3. Electropherograms of 1 mM formate measured at three different extraction times: (a) 60 s,
423
(b) 210 s and (c) 360 s. CE conditions as for Fig. 2. SLM conditions: organic solvent, methanol;
424
donor, 1 mM formate in 100 mM NaCl solution; acceptor, DI water.
d
te
425
M
422
Fig. 4. A. Electropherograms of blank solutions measured after extraction for 60 s: (a) standard
427
solution of 100 mM NaCl, (b) human serum, (c) human whole blood. B. Electropherograms of
428
the human serum sample unspiked (0 mM) and spiked with 1 mM, 10 mM and 20 mM formate
429
measured after extraction for 60 s. CE and SLM conditions as for Fig. 3.
430
Ac ce p
426
431
Fig. 5. Electropherograms of human serum sample of the methanol intoxicated patient after 4 h
432
of hemodialysis. CE measurements were performed with (a) Beckman P/ACE 5000 and (b)
433
Agilent 7100 CE instruments. CE conditions as for Fig. 2; injection, (a) 0.5 psi for 5 s and (b) 50
434
mbar for 5 s; SLM conditions as for Fig. 3; extraction time, 60 s.
21 Page 21 of 29
*Highlights (for review)
ce pt
ed
M
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Sample pretreatment is in-line coupled to commercial CE for automated operation Samples are extracted using disposable devices with supported liquid membranes Formate is determined in undiluted whole blood and serum using CE-UV detection Total analysis time including blood sampling, extraction and analysis is less than 4 min The method might be used for clinical analyses of formate after methanol intoxication
Ac
Page 22 of 29
Figure 1
A
B Electrode
cr
ip t
Capillary
an
us
Cap
d Ac ce pt e
Acceptor SLM Donor PU foam
M
Sample vial
Figure 1 Page 23 of 29
Figure 2
10
8 oxalate
mAU
6
chloride, nitrite, sulfate, nitrate
formate tartrate
ip t
4
2
cr
fumarate
0 1.8
us
1.6
M
an
1.0 1.2 1.4 migration time (min)
d
0.8
Ac ce pt e
0.6
Figure 2 Page 24 of 29
Figure 3
50
chloride
40
20
ip t
formate a
10
b
1.4
1.6
us
0.8 1.0 1.2 migration time (min)
M
an
0.6
d
0.4
cr
c
0
Ac ce pt e
mAU
30
Figure 3 Page 25 of 29
Figure 4
A
chloride
30
mAU
20
10
ip t
formate
0 a
-10
cr
b c
B
0.6
0.8 1.0 1.2 migration time (min)
1.4
1.6
us
0.4
chloride
an
30 formate
20 mM
10
M
mAU
20
d
10 mM 1 mM
Ac ce pt e
0
0 mM
0.4
0.6
0.8 1.0 1.2 migration time (min)
1.4
1.6
Figure 4 Page 26 of 29
Figure 5
80
chloride
formate
60
ip t
40
cr
20 b
0 1.4
1.6
us
0.8 1.0 1.2 migration time (min)
M
an
0.6
d
0.4
Ac ce pt e
mAU
a
Figure 5 Page 27 of 29
Table 1
Table 1. Analytical parameters for the determination of formate using the SLM-CE method. CE conditions as for Fig. 2, SLM conditions as for Fig. 3, extraction time 60 s. n = 5, calibration range = 0.5 – 20 mM.
RSD (%), PA, 1 mM 5.5 6.6 7.7
LOD (µM) 35 30 35
Recovery (%), 1 mM 18 21 19
r2 0.999 0.998 0.998
ip t
Standard solution Human serum Human whole blood
RSD (%), MT, 1 mM 0.5 1.4 1.5
Ac
ce pt
ed
M
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cr
MT: migration time PA: peak area
Page 28 of 29
Table 2
Table 2. Comparison of the proposed method with other methods used for analysis of formate in blood samples.
Sample matrix Plasma
Sample dilution yes
Sample volume 6 µL
Analysis time > 15 mina
LOD
Ref.
152 µM
[32]
HS-GCCommercial FID GC-MS Commercial HS-GC-MS Commercial
Off-line
Whole blood
yes
500 µL
> 40 minb
n.r.
[29]
Off-line Off-line
Whole blood Whole blood
yes yes
200 µL 500 µL
> 90 min n.r.
20 µM [30] c 652 µM [31]
CE-C4D
Lab-made
Off-line
Serum
yes
n.r.
~ 3 mina
220 µM
[34]
µD-CEC4 D SLM-CEUV
Lab-made
In-line
< 4 min
15 µM
[35]
Commercial
In-line
Whole blood, yes serum, plasma Whole blood, no serum
30-35 µM
This work
cr
us
5 µL
ip t
Sample treatment Off-line
Enzymatic
Analytical system Commercial
10 – 15 µL
< 4 min
an
Method
a
– sample pretreatment time is not included – sample pretreatment time only, time of GC-MS analysis is not included c – limit of quantification
M
b
Ac
ce pt
ed
HS-GC-FID – headspace-gas chromatography-flame ionization detection HS-GC-MS – headspace-gas chromatography-mass spectrometry CE-C4D – capillary electrophoresis-capacitively coupled contactless conductivity detection µD – microdialysis
Page 29 of 29
S0021-9673(13)00838-8 http://dx.doi.org/doi:10.1016/j.chroma.2013.05.058 CHROMA 354375
To appear in:
Journal of Chromatography A
Received date: Revised date: Accepted date:
2-4-2013 23-5-2013 24-5-2013
Please cite this article as: P. Pant˚ucˇ kov´a, P. Kub´anˇ , P. Boˇcek, Supported liquid membrane extraction coupled in-line to commercial capillary electrophoresis for rapid determination of formate in undiluted blood samples, Journal of Chromatography A (2013), http://dx.doi.org/10.1016/j.chroma.2013.05.058 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Supported liquid membrane extraction coupled in-line to commercial capillary
2
electrophoresis for rapid determination of formate in undiluted blood samples
3
Pavla Pantůčková, Pavel Kubáň*, Petr Boček
ip t
4 5
Institute of Analytical Chemistry of the Academy of Sciences of the Czech Republic, v. v. i.,
7
Veveří 97, CZ-60200 Brno, Czech Republic
us
cr
6
8
Dr. Pavel Kubáň, e-mail: [email protected], Tel: +420 532290140, Fax: +420 541212113, Institute
10
of Analytical Chemistry of the Academy of Sciences of the Czech Republic, v. v. i., Veveří 97,
11
CZ-60200 Brno, Czech Republic
13
Keywords: blood samples; capillary electrophoresis; formate; in-line sample pretreatment;
14
methanol intoxication; supported liquid membranes
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Page 1 of 29
Abstract
16
A cheap, disposable sample pretreatment device with planar supported liquid membrane (SLM)
17
was proposed, assembled and placed into an autosampler carousel of a commercial capillary
18
electrophoresis (CE) instrument for automated pretreatment and analysis of formate in undiluted
19
whole blood and serum samples. All analytical procedures except for filling the pretreatment
20
device with donor and acceptor solutions, i.e. extraction across SLM, injection of the extracted
21
sample and CE-UV determination of formate, were performed fully automatically. The
22
pretreatment device required only µL volumes of blood sample and organic solvent per
23
extraction and was disposed off after each extraction. Good repeatability of peak areas ( 7.7%)
24
and migration times ( 1.5%), linear relationship (r2 = 0.998 – 0.999) and limits of detection (
25
35 µM) were achieved. The overall analytical process including blood withdrawal, filling the
26
SLM device with respective solutions, extraction of blood sample, injection into separation
27
capillary and CE separation of formate from other anions took less than 4 min. The method was
28
proved useful by direct determination of elevated formate concentrations in undiluted serum
29
samples of a methanol intoxicated patient. Due to its compatibility with currently commercially
30
available CE instrumentation, disposability of extraction devices, minimum sample
31
handling/consumption, and short extraction/analysis times, the developed method might be
32
attractive for rapid diagnosis of methanol poisoning in clinical and toxicological laboratories.
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1. Introduction
34
Clinical samples, such as human serum and whole blood, contain usually limited amount of
35
target analytes and high concentrations of bulk components, which often deteriorate performance
36
of capillary electrophoresis (CE). Most serious complications arise from the presence of major
37
blood components, e.g. proteins, lipids and salts and additionally of red blood cells in the case of
38
whole blood. Proteins, lipids and particulate matter tend to adhere to the inner surface of
39
separation capillaries and have severe effect on CE performance, selectivity and sensitivity [1-4].
40
Blood samples are therefore only scarcely injected without sample pretreatment and in most CE
41
applications they are pretreated in order to eliminate their matrices [5-9]. The pretreatment is
42
normally performed using stand-alone sample pretreatment instrumentation and the cleaned-up
43
and, in most cases, also preconcentrated samples are then off-line transferred to CE for injection.
44
However, the off-line transfer may be inconvenient due to the risk of sample contamination
45
induced by manual handling, loss of analytes and need for unattended operation. Set-ups
46
allowing for direct coupling of pretreatment and analytical techniques are therefore beneficial
47
and reports on direct coupling of various pretreatment devices to CE were comprehensively
48
reviewed by Valcárcel et al. [5,6]. Vast majority of these set-ups was realized with lab-made CE,
49
which enabled the required flexibility for coupling the pretreatment techniques to CE.
50
Contrary to lab-made CE, direct coupling of sample pretreatment techniques to commercial CE
51
necessitates additional alterations of the pretreatment devices in order to enable their
52
compatibility with CE injection systems and/or sample vials/carousels [7]. Direct coupling of
53
sample pretreatment to CE using external instrumentation, such as flow-through programmable
54
arm [10-12] and pump system [13,14], was reported. However, in order to transfer the pretreated
55
samples to CE, the external instrumentation must be sophisticated and must be precisely
Ac ce p
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3 Page 3 of 29
synchronized with the movement of the autosampler carousel. The need for the external
57
instrumentation and its synchronization with CE can be overcome by using sample pretreatment
58
in-capillary [15-17], in-vial [18-21] or at-capillary tip [22,23]. These approaches are particularly
59
interesting since the entire pretreatment, injection and separation process is performed in an
60
automated manner inside the commercial CE. Nevertheless, in-capillary sample pretreatment
61
suffers from the fact that complex samples have to be flushed through the separation capillary
62
during sample loading. To avoid direct contact of their matrix components with the capillary
63
inner walls, samples were usually loaded off-line [15-17]. In-vial arrangement eliminates the
64
contact of the separation capillary with the sample matrix by inserting a supported liquid
65
membrane (SLM), a thin porous polymeric membrane impregnated with water immiscible
66
solvent [24-27], between the aqueous donor and acceptor solution. However, fabrication of
67
SLM-based pretreatment devices, which are compatible with commercial CE instruments, is
68
elaborate and does not allow their disposable use. Regular conditioning of the SLM is therefore
69
necessary and limited life-time of these devices was reported for pretreatment of complex
70
samples [18-20]. The major drawback of the set-up where a small aqueous acceptor droplet is
71
formed at the separation capillary tip and covered by a thin organic layer is limited stability of
72
the organic layer in aqueous solutions [22,23]. Moreover, stability of the entire droplet is reduced
73
when the donor solution is agitated, which is necessary to ensure constant replenishment of
74
analytes at the donor/acceptor interface.
75
In this manuscript, a sample pretreatment device with SLM [21] was in-line coupled to
76
commercial CE for direct determination of formate in blood samples. Formate is the major
77
metabolite of methanol dehydrogenation in human bodies, can be accumulated therein and is
78
primarily responsible for the toxicity in methanol poisonings [28]. Analytical techniques, which
Ac ce p
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4 Page 4 of 29
are currently used for determination of formate in suspected methanol poisonings, are headspace
80
gas chromatography (GC) with flame ionization or mass spectrometric detection [29-31] and
81
enzymatic methods combined with spectrophotometry [32,33]. However, GC methods are time-
82
consuming and require sample derivatization, and enzymatic methods necessitate considerable
83
incubation times and reagents are costly. Two CE methods were reported recently for
84
determination of formate in human blood samples associated with methanol poisonings, which
85
offer reduced analysis times and costs [34,35]. Note however, that lab-made CE instrumentation,
86
which must be manually operated by trained personnel and is not acceptable for routine use in
87
clinical laboratories, was used in these reports and formate was detected with conductivity
88
detectors, which are not standard equipment in commercial CE systems. Determination of
89
formate was performed in blood serum [34], which additionally necessitates whole blood
90
clotting, centrifugation and serum collection prior to analysis and therefore considerably
91
increases the total analysis time. Microdialysis membranes and units, which are rather expensive
92
and cannot be used as disposable extraction devices, were used for sample pretreatment in [35].
93
Moreover, additional sample handling, i.e. 100-fold [34] and 10-fold [35] dilution, was required
94
in order to minimize the effect of sample matrix and to reduce sample consumption.
95
In this report, a new method was developed for direct analysis of formate in µL volumes of
96
undiluted blood samples based on in-line coupling of disposable sample pretreatment device to
97
commercial CE equipped with standard UV-Vis detector. The overall analysis time, including
98
whole blood withdrawal and automated sample extraction, injection, CE separation and
99
quantification of formate, was less than 4 min. Beckman P/ACE 5000 series as well as Agilent
100
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7100 CE instruments were proved useful for direct analysis of formate in raw blood samples.
101
5 Page 5 of 29
2. Materials and methods
103
2.1. Instrumentation
104
2.1.1. Sample pretreatment devices
105
A sample pretreatment device, which is compatible with injection system of Beckman P/ACE
106
5000 series CE instruments (Beckman Instruments, Fullerton, CA, USA), and its application in
107
pretreatment of complex samples was described in detail previously [21]. Volume of acceptor
108
and donor solution was 15 L, volume of organic solvent for SLM impregnation was 10 L and
109
complete pretreatment device was disposed off after each extraction in the actual measurements.
110
Optimization of the analytical method for direct coupling of SLM extractions to commercial CE
111
for formate analyses was performed with the Beckman P/ACE CE instrument.
112
The currently most popular commercially available CE system (7100 CE, Agilent Technologies,
113
Waldbronn, Germany) was also used for direct coupling to SLM extractions. Volume and size of
114
sample vials are reduced in Agilent 7100 CE compared to Beckman P/ACE 5000 instruments
115
and the SLM device had to be down-scaled accordingly (see Fig. 1). Donor and acceptor units
116
were pre-cut from 200 µL polypropylene (PP) micropipette tips (Kartell Spa, Noviglio, Italy) to
117
fit the internal diameter of the Agilent sample vials (Part No. 5182-0567). PP membranes
118
(Accurel PP 1E R/P, Membrana, Wuppertal, Germany, 100 µm thick, average pore size 0.1 µm)
119
were pre-cut from the Accurel PP sheet using a 11 mm cork borer. SLM devices were assembled
120
according to the following procedure, which was performed routinely in ca. 1 min. First, the PP
121
membrane was impregnated with 10 L of an organic solvent. Second, the membrane was placed
122
on top of the donor unit and firmly pressed against the bottom of the acceptor unit. Third, the
123
donor and acceptor unit was filled with 10 µL of donor and acceptor solution, respectively, and
124
measurement of extraction time was started. Note that the units are filled only partially and
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6 Page 6 of 29
empty space was left below and above the donor and acceptor solution, respectively (Fig. 1), for
126
ease of manipulation. Fourth, the SLM device was accommodated in the sample vial, which was
127
placed into CE autosampler. The issue of prefabrication and long-term storage of SLM devices
128
with impregnated membranes was not examined as it is beyond the scientific scope of this
129
manuscript.
130
In addition to reduced size of sample vials, geometry of the HV electrode and its position relative
131
to the injection end of the separation capillary is different in Agilent CE instruments. The
132
capillary is placed in centre of a tubular HV electrode and the capillary tip and electrode rim are
133
positioned close to the vial bottom during injection (see Fig. 1B). For this reason, a 5 3 mm
134
cylinder was cut from soft polyurethane (PU) foam (unknown producer) and placed at the sample
135
vial bottom below the pretreatment device. During sample injection, the sample vial is lifted up
136
and the injection end of the separation capillary touches the SLM. As the SLM device is
137
positioned on the soft PU foam, the device can be easily pressed down into the foam (see Fig.
138
1B), which releases the pressure induced during the upward movement of the sample vial and
139
avoids perforation of the SLM. Moreover, it simultaneously ensures that the injection end
140
remains in direct contact with the SLM during injection. The injection end of the separation
141
capillary overlaps the rim of the tubular HV electrode by ca. 0.5 mm (see Fig. 1). This position
142
differs from that previously optimized and used for Beckman P/ACE instruments. The reason is
143
better repeatability of injections compared to those observed when the capillary tip and the
144
electrode rim were levelled off or when the electrode rim overlapped the capillary tip.
145
After extraction, the pretreatment device was removed from the sample vial and was disposed
146
off. A new set of donor/acceptor units and a new SLM was used for each extraction. The analyte
147
transfer across the SLM, expressed as recovery, was calculated as described previously [36].
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2.1.2. Capillary electrophoresis
150
Automated CE instruments P/ACE 5000 (Beckman Instruments) and 7100 CE (Agilent
151
Technologies) equipped with UV-Vis detector (operated at 214 nm in indirect mode) were used.
152
Indirect absorbance signals were software-wise inverted in order to obtain positive peaks.
153
Electrophoretic separations using P/ACE instrument were performed in a fused-silica (FS)
154
capillary (75 µm ID/365 µm OD, 27/20 cm total/effective length, Polymicro Technologies,
155
Phoenix, AZ, USA). Prior to the first use, the bare capillary was treated as described recently
156
[21]. Between two CE runs, the capillary was rinsed with DI water and BGE solution at 20 psi
157
(1379 mbar) for 18 s each. Anionic analytes were analyzed in anionic mode without reversal of
158
EOF, i.e. with cathode at the injection side. CE separations were performed at a constant voltage
159
of – 30 kV. Capillary temperature was maintained at 25°C and injections were performed
160
hydrodynamically at 0.5 psi (34.5 mbar) for 5 s. The CE instrument was controlled and analytical
161
signals were processed using Beckman P/ACE Station Version 1.0 software.
162
The Agilent 7100 CE system was operated at a potential of – 30 kV applied at the injection side
163
of the separation capillary for all runs. Separation capillary used was a FS capillary (75 µm
164
ID/375 µm OD, 31/22.5 cm total/effective length, Polymicro Technologies). Conditioning of the
165
bare capillary and rinsing procedure between two CE runs were performed according to the
166
procedure described above at a pressure of 950 mbar. Capillary temperature was maintained at
167
25°C and injections were performed hydrodynamically at 50 mbar for 5 s. The CE instrument
168
was controlled and analytical signals were acquired by ChemStation CE software.
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169 170
2.2. Reagents, BGE solutions, standards and blood samples
8 Page 8 of 29
All chemicals (reagent grade) were purchased from Pliva-Lachema, Brno, Czech Republic;
172
Sigma, Steinheim, Germany; and Fluka, Buchs, Switzerland. DI water with resistivity higher
173
than 18 Mcm was prepared by exchange of ions in a mixed-bed ion exchanger water
174
purification system G 7749 (Miele, Gütersloh, Germany). 1 M stock solution of Cl- was prepared
175
from NaCl. Stock solutions of other inorganic anions (100 mM) were prepared from their Na+ or
176
K+ salts. Stock solutions of organic anions (100 mM) were prepared from their Na+ or K+ salts or
177
from organic acids. All stock solutions were kept refrigerated at 4C. Standard sample solutions
178
of the inorganic and organic anions were freshly prepared from these stock solutions and were
179
diluted with DI water. Human serum albumin (HSA) was purchased from Sigma. BGE solutions
180
for CE measurements were prepared from DL-mandelic acid (Sigma) and -aminocaproic acid
181
(Pliva-Lachema) and were kept refrigerated at 4C. The optimum separation of formate from
182
other inorganic/organic anions was achieved in BGE solution consisting of 15 mM DL-mandelic
183
acid and 25 mM -aminocaproic acid (pH 4.35). SLMs were prepared by impregnating PP
184
membranes (Membrana) with methanol, 1-butanol and 1-pentanol (Fluka). Human serum and
185
whole blood samples were collected from healthy volunteers at the Institute of Analytical
186
Chemistry; a written informed consent was signed by the volunteers before the experiments.
187
Serum samples were prepared from clotted intravenous blood by centrifugation at 6000 rpm for
188
10 min. Whole blood samples were collected either from a finger-stick (for instant analysis) or as
189
anti-coagulated (EDTA addition) intravenous blood. Serum samples of a patient diagnosed and
190
treated with acute methanol poisoning were donated by Prof. Robert Bocek from Department of
191
Anaesthesiology, Resuscitation and Intensive Care, Hospital and Policlinics Havířov, Czech
192
Republic. All blood samples were used undiluted, stored at –20°C and allowed to warm up to the
193
ambient temperature prior to analysis.
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3. Results and discussion
196
3.1. BGE solution
197
Basic configuration of most commercially available CE instruments consists of a CE module and
198
a UV-Vis detector. The selection of BGE solutions for analysis of formate in blood samples was
199
therefore optimized with respect to detection in UV-Vis region. Formate posses no chromophore
200
and is therefore detected in indirect mode using BGE solutions composed of highly UV-
201
absorbing species. A BGE solution consisting of DL-mandelic and -aminocaproic acid was
202
shown suitable for CE-indirect UV determination of various inorganic and organic anions
203
previously [37] and was adopted for our measurements. CE separations of formate from other
204
anions, which might be present in blood samples and might interfere with formate determination,
205
i.e., chloride, sulfate, nitrite, nitrate, oxalate, tartrate, fumarate, citrate, maleate, malate,
206
malonate, lactate, carbonate, phosphate and acetate, were predicted in various BGE solutions by
207
using a modelling freeware Peakmaster [38]. BGE solutions were theoretically examined in the
208
range of 5-15 mM for DL-mandelic acid and of 6-60 mM for -aminocaproic acid (pH 4 – 4.9).
209
CE separations in selected BGE solutions were subsequently verified experimentally. Due to the
210
low pH values of the examined BGE solutions, reversal of EOF was not required and fast
211
separations of most analytes were achieved in counter-electroosmotic separation mode.
212
Separation of a standard solution of inorganic and organic anions in the optimum BGE solution
213
(15 mM DL-mandelic acid and 25 mM -aminocaproic acid at pH 4.35) is shown in Fig. 2.
214
Formate is baseline separated from the fast inorganic anions (chloride, sulfate, nitrite and
215
nitrate), and from oxalate, tartrate and fumarate. All other anions from the above mentioned list
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216
exhibited even slower migration than fumarate, did not interfere with formate determination, and
217
were detected at times longer than 1.8 min.
218
3.2. Donor and acceptor solution
220
Donor solutions were standard solutions of respective analytes and NaCl, blood serum and whole
221
blood. The actual composition of standard solutions is mentioned in the relevant text further on.
222
In all cases, donor solutions were used undiluted and untreated. This minimizes the possibility
223
for sample contamination, which might be implied by sample dilution and/or addition of
224
reagents, and ensures very fast sample handling and sufficient sensitivity of the analytical
225
method. Acceptor solutions were pure DI water. It has been described previously that for
226
extractions performed directly at the SLM surface, pH gradients across the SLM offer no
227
advantage over the simplest possible set-up with pure DI water as acceptor solution [21,36].
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3.3. Organic solvent used in SLM
230
Extractions of small inorganic and organic anions across SLMs formed by impregnation of PP
231
membranes with short-chain aliphatic alcohols were reported previously [39-41]. Methanol, 1-
232
butanol and 1-pentanol were therefore examined as organic solvents in SLMs. Recoveries after
233
60 s extraction of 1 mM formate in a standard solution of 100 mM NaCl were approximately
234
18%, 4% and 6% for methanol, 1-butanol and 1-pentanol, respectively. In addition to the SLMs,
235
dry PP membrane and a 500 Da molecular weight cut-off dialysis membrane [35] were
236
examined. Formate was not transferred across the dry PP membrane as the hydrophobic
237
membrane completely separated the donor and acceptor solution and ion transfer between the
238
two solutions was not feasible. Transfer of formate across the dialysis membrane was
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11 Page 11 of 29
comparable to that across the SLM impregnated with methanol, however, at the expense of
240
increased chloride transfer and ca. 1000-fold higher costs of the membrane.
241
Repeatability of the formate transfer was therefore further examined across the SLMs only. Most
242
repeatable results were observed for methanol, which is consistent with the previous findings
243
[40]. The alcohols are evenly distributed in the pores and on the surface of the PP membrane
244
during the SLM impregnation. Nevertheless, as the exceeding organic solvent partially
245
evaporates from the membrane surface during manipulation with the SLM, the thickness and
246
uniformity of the liquid membrane, which are responsible for the analyte transfer across SLMs,
247
vary for individual solvents. Consequently, this has a direct bearing on the extraction
248
performance and influences the total transfer (expressed as recoveries) and transfer repeatability.
249
Best extraction performance achieved for SLMs impregnated with methanol may be explained
250
by the fact that methanol evaporates most rapidly from the SLM surface and remains only in the
251
membrane pores, which ensures rather constant SLM properties. Methanol was therefore applied
252
as the liquid membrane in all subsequent SLM extractions.
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3.4. Extraction time
255
It has been shown recently that minute extraction times can be achieved for stagnant diffusion
256
when the diffusive layer is minimized and pretreated samples are injected directly from the SLM
257
surface [36]. For lab-made CE systems the extraction and injection process can be synchronized
258
and instant CE injection can be performed almost at the same time when the pretreatment device
259
is assembled [41]. This is, however, not possible in commercial CE instruments and certain time
260
period is required to perform CE initialization, flushing and injection after assembling the SLM
261
device. The minimum extraction time was set at 60 s and time program of the CE system was
12 Page 12 of 29
adjusted in order to perform the first injection 60 s after the SLM device was filled with donor
263
and acceptor solutions. The SLM device was then retained in the autosampler carousel and
264
subsequent injections were performed from the device at 150 s intervals. Fig. 3 depicts analysis
265
of a standard solution of 1 mM formate and 100 mM NaCl extracted for 60, 210 and 360 s.
266
Consistent results were achieved for peak areas of formate injected at extraction times up to 10
267
min and gradual decrease was observed for subsequent injections at extraction times of 20 and
268
30 min. The lower analytical signals are likely to be caused by gradual dissolution of the liquid
269
membrane, which may result in considerable changes and dilution of the aqueous solution and
270
analyte concentration at the injection (acceptor) side of the SLM. Maximum recovery was
271
obtained already for the first measurement (60 s extraction time) and no further increase of the
272
formate recovery was observed for subsequent measurements. These experiments demonstrate
273
that the extraction process at the SLM surface has reached equilibrium within the first 60 s.
274
Extraction time of 60 s was used for all subsequent extractions.
275
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3.5. Interferences
277
Composition of human blood is rather consistent and variations in concentrations of most blood
278
constituents are less than 10% in healthy individuals [42]. Human blood consists of major (e.g.
279
HSA, Na+ and Cl-) as well as minor components and effect of the major ones on the extraction
280
performance was investigated. Standard solutions of 1 mM formate were prepared in DI water
281
containing HSA (70 g/L) and various concentrations of NaCl (10 – 100 mM). Performance of the
282
CE separation was not affected by the presence of HSA in donor solutions, which confirms the
283
generally known fact that liquid membranes can efficiently eliminate large molecules, such as
284
proteins. Addition of increasing concentrations of NaCl showed measurable changes in formate
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13 Page 13 of 29
recoveries, which gradually decreased from 35% (10 mM NaCl) to 18% (100 mM NaCl). This is,
286
however, not surprising since the transfer of analyte ions across the methanol-based SLMs
287
depends on concentration ratios of analyte/matrix ions [40]. A gradual increase of chloride peak
288
was also observed during these experiments, demonstrating higher transfer of NaCl due to its
289
elevated initial concentrations. Recovery values for extractions of standard solutions consisting
290
of 90 and 110 mM NaCl (typical range for chloride in blood) and for extractions of undiluted
291
serum and whole blood samples, all spiked with 1 mM formate, ranged between 17 and 21%.
292
Obviously, the variations of the NaCl concentrations in real blood samples do not interfere with
293
sufficient repeatability of the formate recoveries. Hence, the analytical method is well applicable
294
to extractions and analyses of formate in blood samples.
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3.6. Analysis of human blood samples and analytical parameters of the method
297
Electropherograms of blank samples (100 mM NaCl, human serum and human whole blood from
298
a finger-stick) measured after extraction for 60 s are depicted in Fig. 4A. Analytical signals of
299
formate were below the limit of detection of the method and fall within the reference range (0 –
300
400 µM) reported in healthy individuals [43,44]. The blood samples were subsequently spiked
301
with 1, 10 and 20 mM of formate and gradual increase of the formate peak was observed as is
302
depicted for human serum in Fig. 4B.
303
Table 1 summarizes analytical parameters of the presented SLM-CE method. Standard solution
304
of 100 mM NaCl, blood serum and whole blood, all spiked with 1 mM formate, were used for
305
repeatability measurements. RSD values of peak areas of 5 independent extractions were within
306
the range 5.5 – 7.7% and average recoveries ranged between 18% and 21%. Inter-day and intra-
307
day repeatability of peak areas was below 7.7% and 11.8%, respectively. Calibration curves were
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14 Page 14 of 29
plotted for the standard solution consisting of 100 mM NaCl and blood samples, which were all
309
spiked with formate concentrations in 0.5 – 20 mM range. Calibrations were linear in the whole
310
concentration range with correlation coefficients between 0.998 and 0.999. Limits of detection
311
(LODs), defined as 3 S/N ratio, were between 30 – 35 µM for the three different matrices. Note,
312
that even though the method recoveries are relatively low, its sensitivity is sufficient since the
313
LODs are about 2-3 orders of magnitude below the formate concentrations associated with
314
methanol intoxications.
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315
3.7. Analysis of human serum of a methanol intoxicated patient
317
Serum samples of a patient diagnosed with acute methanol poisoning, which were withdrawn
318
after 3 and 4 h of hemodialytic treatment, were analyzed. Concentrations of formate determined
319
in these samples were 12.3 ± 0.5 mM and 10.4 ± 0.4 mM, respectively, and correlate well with
320
the previous results on determination of formate in the same serum samples using CE-C4D [34]
321
and microelectrodialysis-CE-C4D [35]. The elevated formate concentrations in the early stage of
322
the treatment demonstrate serious methanol intoxication of the patient. The subsequent decrease
323
(between 3 and 4 h of treatment) follows the formate half-life in serum under hemodialytic
324
treatment [45]. Fig. 5 depicts analysis of the patient’s serum sample after 4 h of hemodialytic
325
treatment using the P/ACE 5000 series CE (trace a) and Agilent 7100 CE (trace b). Apparently,
326
there are only insignificant differences between the two records and the method can easily, with
327
just slight alterations to the SLM device design, be implemented into various commercial CE
328
instruments.
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4. Conclusions
15 Page 15 of 29
Disposable sample pretreatment devices compatible with injection systems of commercial CE
332
instruments were proposed, assembled and applied to rapid CE-UV analyses of formate in
333
human whole blood and serum. SLM was sandwiched between two conical segments of the
334
pretreatment device and formed a selective barrier between donor and acceptor solutions.
335
Equilibrium between the formate concentrations at the SLM surface was established rapidly and
336
extracted formate was injected into CE separation capillary directly from the acceptor side of the
337
SLM. Stable CE performance was achieved since the SLM eliminated high molecular mass
338
matrix components and enabled only transfer of small molecules. The developed method may
339
find wide application in clinical and toxicological laboratories for the following reasons. The
340
complete extraction process, injection of extracted sample and CE-UV analysis of extracted
341
analytes
342
instrumentation. The method is applied to analysis of few µL of undiluted whole blood and no
343
additional sample handling, such as blood clotting, centrifugation, serum collection and dilution,
344
is necessary. Cost of the pretreatment device is negligible (less then 0.02 € per device) and the
345
entire pretreatment device is disposed off after sample extraction. Total analysis time including
346
whole blood withdrawal, sample extraction, and CE injection/separation/quantification of
347
formate is less than 4 min. Characteristics of the developed method offer an unrivalled analytical
348
performance for formate analyses as can be seen in Table 2.
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using
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performed
commercially
available
analytical
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350
Acknowledgments
351
Financial support from the Academy of Sciences of the Czech Republic (Institute Research
352
Funding RVO:68081715) and the Grant Agency of the Czech Republic (Grant No. 13-05762S) is
353
gratefully acknowledged. Prof. Robert Bocek (Department of Anaesthesiology, Resuscitation
16 Page 16 of 29
354
and Intensive Care, Hospital and Policlinics Havířov, Czech Republic) and Dr. Petr Kubáň
355
(CEITEC, Masaryk University, Brno, Czech Republic) are also acknowledged for donation of
356
serum samples of a patient treated with acute methanol poisoning.
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Figure captions
413
Fig. 1. SLM pretreatment device coupled directly to Agilent 7100 CE instrument. (A) sample
414
vial before sample injection, (B) sample vial during injection of the pretreated sample.
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415
Fig. 2. Electropherogram of a standard solution of anionic analytes. CE measurements were
417
performed with the P/ACE 5000 CE instrument. CE conditions: BGE solution; 15 mM DL-
418
mandelic acid and 25 mM -aminocaproic acid at pH 4.35, voltage, –30 kV; injection, 0.5 psi for
419
5 s. Standard solution contained 0.2 mM chloride, 0.05 mM sulfate, 0.5 mM oxalate, 0.1 mM
420
nitrite, nitrate, formate, fumarate and tartrate.
an
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421
Fig. 3. Electropherograms of 1 mM formate measured at three different extraction times: (a) 60 s,
423
(b) 210 s and (c) 360 s. CE conditions as for Fig. 2. SLM conditions: organic solvent, methanol;
424
donor, 1 mM formate in 100 mM NaCl solution; acceptor, DI water.
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425
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422
Fig. 4. A. Electropherograms of blank solutions measured after extraction for 60 s: (a) standard
427
solution of 100 mM NaCl, (b) human serum, (c) human whole blood. B. Electropherograms of
428
the human serum sample unspiked (0 mM) and spiked with 1 mM, 10 mM and 20 mM formate
429
measured after extraction for 60 s. CE and SLM conditions as for Fig. 3.
430
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431
Fig. 5. Electropherograms of human serum sample of the methanol intoxicated patient after 4 h
432
of hemodialysis. CE measurements were performed with (a) Beckman P/ACE 5000 and (b)
433
Agilent 7100 CE instruments. CE conditions as for Fig. 2; injection, (a) 0.5 psi for 5 s and (b) 50
434
mbar for 5 s; SLM conditions as for Fig. 3; extraction time, 60 s.
21 Page 21 of 29
*Highlights (for review)
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cr
ip t
Sample pretreatment is in-line coupled to commercial CE for automated operation Samples are extracted using disposable devices with supported liquid membranes Formate is determined in undiluted whole blood and serum using CE-UV detection Total analysis time including blood sampling, extraction and analysis is less than 4 min The method might be used for clinical analyses of formate after methanol intoxication
Ac
Page 22 of 29
Figure 1
A
B Electrode
cr
ip t
Capillary
an
us
Cap
d Ac ce pt e
Acceptor SLM Donor PU foam
M
Sample vial
Figure 1 Page 23 of 29
Figure 2
10
8 oxalate
mAU
6
chloride, nitrite, sulfate, nitrate
formate tartrate
ip t
4
2
cr
fumarate
0 1.8
us
1.6
M
an
1.0 1.2 1.4 migration time (min)
d
0.8
Ac ce pt e
0.6
Figure 2 Page 24 of 29
Figure 3
50
chloride
40
20
ip t
formate a
10
b
1.4
1.6
us
0.8 1.0 1.2 migration time (min)
M
an
0.6
d
0.4
cr
c
0
Ac ce pt e
mAU
30
Figure 3 Page 25 of 29
Figure 4
A
chloride
30
mAU
20
10
ip t
formate
0 a
-10
cr
b c
B
0.6
0.8 1.0 1.2 migration time (min)
1.4
1.6
us
0.4
chloride
an
30 formate
20 mM
10
M
mAU
20
d
10 mM 1 mM
Ac ce pt e
0
0 mM
0.4
0.6
0.8 1.0 1.2 migration time (min)
1.4
1.6
Figure 4 Page 26 of 29
Figure 5
80
chloride
formate
60
ip t
40
cr
20 b
0 1.4
1.6
us
0.8 1.0 1.2 migration time (min)
M
an
0.6
d
0.4
Ac ce pt e
mAU
a
Figure 5 Page 27 of 29
Table 1
Table 1. Analytical parameters for the determination of formate using the SLM-CE method. CE conditions as for Fig. 2, SLM conditions as for Fig. 3, extraction time 60 s. n = 5, calibration range = 0.5 – 20 mM.
RSD (%), PA, 1 mM 5.5 6.6 7.7
LOD (µM) 35 30 35
Recovery (%), 1 mM 18 21 19
r2 0.999 0.998 0.998
ip t
Standard solution Human serum Human whole blood
RSD (%), MT, 1 mM 0.5 1.4 1.5
Ac
ce pt
ed
M
an
us
cr
MT: migration time PA: peak area
Page 28 of 29
Table 2
Table 2. Comparison of the proposed method with other methods used for analysis of formate in blood samples.
Sample matrix Plasma
Sample dilution yes
Sample volume 6 µL
Analysis time > 15 mina
LOD
Ref.
152 µM
[32]
HS-GCCommercial FID GC-MS Commercial HS-GC-MS Commercial
Off-line
Whole blood
yes
500 µL
> 40 minb
n.r.
[29]
Off-line Off-line
Whole blood Whole blood
yes yes
200 µL 500 µL
> 90 min n.r.
20 µM [30] c 652 µM [31]
CE-C4D
Lab-made
Off-line
Serum
yes
n.r.
~ 3 mina
220 µM
[34]
µD-CEC4 D SLM-CEUV
Lab-made
In-line
< 4 min
15 µM
[35]
Commercial
In-line
Whole blood, yes serum, plasma Whole blood, no serum
30-35 µM
This work
cr
us
5 µL
ip t
Sample treatment Off-line
Enzymatic
Analytical system Commercial
10 – 15 µL
< 4 min
an
Method
a
– sample pretreatment time is not included – sample pretreatment time only, time of GC-MS analysis is not included c – limit of quantification
M
b
Ac
ce pt
ed
HS-GC-FID – headspace-gas chromatography-flame ionization detection HS-GC-MS – headspace-gas chromatography-mass spectrometry CE-C4D – capillary electrophoresis-capacitively coupled contactless conductivity detection µD – microdialysis
Page 29 of 29