focus on Mass Spectrometry &


Electrospray as an atmospheric pressure ionization technique has become a significant contribution to analytical chemistry when coupled with modern mass spectrometry. Although an early report of electrospray determination of industrial and biopolymers is what originally generated considerable interest in the technique [1] [2], it was the on-line coupling of HPLC with electrospray mass spectrometry (LC/MS) [3] as well as tandem mass spectrometry (LC/MS/MS) [4] that really propelled the interest and commercial development of this technology [5].


As a result we are all indebted to the late Professor John Fenn for his major contributions that have lead to an exciting addition to our analytical tool box. There are now thousands of publications describing a wide variety of impressive applications ranging from the qualitative characterisation of trace unknown, previously intractable, compounds to the widespread selected reaction monitoring (SRM) LC/MS determination of multi-residue substances in biological, environmental, and industrial samples.


In this report we describe an important modern extension of electrospray mass spectrometry employing microfluidics and chip-based devices that employ nano electrospray flows more than 1000-times lower than conventional electrospray procedures. This technology could potentially lend itself to lab-on-a-chip (LOC) devices in the future [6] as well as some noteworthy analytical benefits including improved analyte sensitivity, normalised response of related analytes and reduced ion suppression by matrix interferences [7]. In some instances, although counter intuitive at first thought, it is possible to analyse biological extracts by nanoESI via simple infusion sample introduction in lieu of HPLC sample separation [8]. This approach can lead to faster sample throughput in the absence of the often more time-consuming HPLC chromatographic separation process.


As an example of this latter concept we describe an automated procedure for the on-line sequential extraction of dried blood spots (DBS) coupled with chip-based infusion nanoESI analysis of the DBS extract. The subject of DBS, or dried ‘matrix’ spots (DXS) with ‘X’ being any sample matrix which is relevant to this format, is a topic of considerable current interest [9]. Traditionally DBS samples are used, for example, in newborn screening [10] and more recently in preclinical pharmaceutical drug discovery [9].


The strategy described in this report employs a robotic platform directly coupled to any modern atmospheric pressure ionisation (API) mass spectrometer system. The TriVersa NanoMate robot houses an ANSI (American National Standards Institute) format plate, which can accommodate a DBS card containing applied biological samples. The latter can range from whole blood, as described in this report, to plasma, urine, bile or a wide variety of other biological samples. The robot is equipped with a mandrel that can pick up a pipette tip containing a few microlitres of extraction/spray solvent, which extracts analytes from the sample surface via a micro liquid junction between the pipette tip and the DBS surface. By a repetitive dispense-aspirate procedure drug and metabolite residues contained on the DBS card surface are extracted into the pipette tip, which is then delivered to a microfabricated chip. At this point nanoESI commences to provide infusion electrospray mass spectra for the components extracted from the DBS card. This process is called liquid extraction surface analysis (LESA) and was first described by Kertesz et al. [11] [12] and more recently by Hooper et al. [13].


Here, we describe a semi-automated approach for a simple and effective way to automate the bioanalytical determination of drugs in DBS samples without the need to punch spots from the DBS substrate or application of surface tension modifiers. This work was first reported at the 58th Conference on Mass Spectrometry and Allied Topics [14].


Spectroscopy


Automated Liquid Extraction Surface Analysis (LESA) from a Dried Blood Spot Card Holder via Chip-Based Nanoelectrospray


Geoffrey Rule, Daniel Eikel, Jason Vega, Chris Alpha and Jack Henion* Advion BioSciences, Inc, 19 Brown Rd, Ithaca, NY 14850


This report describes a novel dried blood spot (DBS) card holder equipped with four defined surface extraction areas suitable for automated liquid extraction surface analysis (LESA) of dried biological sample spots. In brief, a commercially available DBS card containing four spotted whole blood samples is manually inserted into the card holder while a hinged cover containing a sequence of four circular ridges is clamped directly onto the sample substrate surface to define four constrained surface extraction regions for each sample. The LESA technique is then implemented by sequentially delivering a pipette tip containing a sample extraction/electrospray solvent to the sample surface. Analytes extracted from the DBS sample are then analysed directly by infusion nanoelectrospray mass spectrometry. Amlodipine and its tetradeuterated internal standard produced a linear calibration curve from 10 ng/mL to 10,000 ng/mL from fortified human whole blood with acceptable accuracy and precision using this approach.


Materials & Methods


DBS sample preparation Control human, lithium heparin, whole blood (Bioreclamation, NY) was fortified with amlodipine across the concentration range from 10-10,000 ng/mL along with its tetra deuterated stable isotope internal standard fortified at the 4000 ng/mL level in each sample. Amlodipine was purchased as its besylate salt and reference standard (US Pharmacopeia, MD) and amlodipine-D4 was purchased as is maleic acid partner (Toronto Research Chemicals, ON). The chemical structures for these compounds are shown in Figure 1. Stock solutions (1 mg/mL) were prepared in 50/50 (v/v) methanol/water. The whole blood was mixed with the analyte stock solutions prior to spotting the paper blue striped FTA DMPK-C substrate cards (GE Healthcare/Whatman, NJ) to obtain the required amlodipine (10 ng/mL to 10,000 ng/mL) and D4 amlodipine (4000 ng/mL) concentrations. Aliquots (12 microlitres) of the fortified human whole blood samples were manually spotted onto paper cards via a pipette and air dried for 2h at room temperature.


DBS Card Holder


Since it can be difficult to form a micro liquid junction between the pipette tip and a porous surface such as a cellulose-based DBS card, a special DBS card holder was fabricated in-house, from aluminum, to provide a means for solvent extraction of the DBS card from a defined region of the DBS sample. This was accomplished by installing four stainless steel (or peek) fittings (Idex, Inc, Bristol, CT 06010) with an inside diameter of 3mm in the hinged cover of the card holder and spaced to be centered over each of four dried blood spot sample locations. When the cover of the DBS card holder is lowered onto the base structure, the latter has a ridged surface to accommodate the rim of the mating fitting thus creating a tight, sealed region to confine dispensed extraction solvent applied to the DBS card surface (Figure 2).


The card may be placed between the base and cover of this device such that the standard four circled regions, containing dried blood spot samples, are centered with respect to the fittings (Figure 3). The hinged cover is then closed and clamped tightly onto the four DBS sample areas. As suggested in Figure 2 the rim of the fittings create a sealed region in the centre of the applied dried blood spots which confines the solvent extraction region to a defined area of about 3mm diameter (vide infra). This allows the selected extraction-spray solvent introduced by the pipette tip to be distributed to a confined cross sectional area of the dried blood spot. This area is created by the perimeter of the fitting as it is compressed upon the dried blood spot sample.


The extraction-spray solvent for this application was 70% methanol, 30% water, 0.1% formic acid. It should be noted that the selection of this solvent should be predicated by a combination of appropriate solvent strength for optimal extraction of the target analytes at the expense of endogenous components as well as the appropriate nanoESI spray characteristics.


Figure 1. Structures of amlodipine and its tetradeuterated internal standard


INTERNATIONAL LABMATE - JANUARY/FEBRUARY 2012


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