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Immobilized Liquid Extraction™ of Chlorphenaramine Directly from Undiluted SerumIntroduction The following application note demonstrates the ability of Immobilized Liquid Extraction 96-Well Plates to rapidly extract chlorpheniramine directly from undiluted serum samples. Chlorpheniramine, often referred to as its salt chlorphenamine maleate, is a first-generation antihistamine used in the prevention of the symptoms of allergic conditions such as rhinitis and urticaria. ILE Well Plates provided rapid extractions of chlorpheniramine directly from non-diluted serum samples of volumes of 50 to 200 microliters with both high efficiency (94.0-98.2%) and precision (3.3-4.7% RSD). Immobilized Liquid Extraction (ILE) 96-Well Plates provide analysts reliable, rapid and environmentally friendly high-throughput extractions without protein crashing, sample dilution, emulsion formation, solvent exchange/reconstitution or dry wells. ILE Well Plates involve minimal user management, especially on a well-to-well basis, do not require vacuum or pressure systems, and are capable of extracting small molecules directly from blood, serum or any aqueous biological matrix. Materials and Method Samples: All samples consisted of either 50uL or 100uL goat serum (Midland Bio Products). The 100uL samples were undiluted, while the 50uL serum samples were diluted 1:1 with water to 100uL. All samples were adjusted to pH 11 with 10.0 M NaOH. Samples and non-extracted calibration spikes were created appropriately at a concentration of 2500 ng/mL using certified analytical standards (Cerilliant Corporation). Extraction Media: All extractions were performed using the Immobilized Liquid Extraction™ 96-Well Plates with a trifluoropropylmethylsiloxane extracting phase. Each well of an ILE Well Plate is coated with a thin layer of polymer (immobilized phase) that acts as the extracting solvent. The trifluoropropyl extracting phase used in this experiment provides enhanced selectivity for extracting compounds with lone pairs of electrons. Back-Extraction Solvent: J.T. Baker Baker Analyzed® Acetonitrile (HPLC Grade). GC-MS Conditions: HP-5971 GC-MS in SIM mode, 1µl splitless injection valve, on a 30m x 0.25mm i.d. X 0.25µm VB-5 column. Method: Samples were extracted using the ILE method (see Figure 1). Namely, a sample is directly exposed to the polymer for a period of time, allowing targeted analytes partition between the aqueous sample and the immobilized phase. Compounds which partition into the immobilized solvent are then back-extracted (eluted) into a small amount of appropriate LC or GC solvent. Figure 1 Results and Discussion Extraction Time, Efficiency and Reproducibility: Extraction efficiency was investigated by extracting a known quantity (2,500 ng/mL) of chlorpheniramine from 100uL samples. The MS response given for each extracted sample was compared to the MS response of an unextracted standard that represents 100% recovery. Two series of 100 uL serum samples (n=7) were extracted for ten and twenty minutes, respectively to determine required extraction time. At ten minutes, an absolute extraction efficiency of 71.4% (5.7% RSD) was achieved, while a twenty minute extraction yielded an efficiency of 94.3% (4.3% RSD). Figure 2 displays the extraction efficiency of chlorpheniramine from a 100uL serum sample as a function of time. Figure 2 Direct Serum Extraction: Many extraction techniques require the dilution of a sample to ensure a rapid, efficient, and reproducible extraction. Consequently, a sample with a volume which nears the capacity of the extraction device may not be diluted such that the resulting sample dilution is of a volume greater than the capacity of the extraction device. ILE 96-Well Plates are able to extract directly from serum with negligible effects on extraction time, efficiency and reproducibility. A series of experiments was conducted to show this characteristic.
This experiment compared two data sets of 100 uL samples (n=7). The first set consisted of 100 uL serum sample spiked with 2,500 ng/mL chlorpheniramine, adjusted to pH 11 with 10.0N NaOH. The second set of samples consisted of 50 uL serum, diluted 1:1 to 100 uL with water, adjusted to pH 11 with 10.0N NaOH. The second set of samples was spiked such that the 100 uL diluted sample had a 2,500 ng/mL concentration of chlorpheniramine. Each sample was extracted for twenty minutes, which had previously been determined as a sufficient amount of time.
Extraction efficiency and reproducibility were measured under each set of conditions. The pure serum sample exhibited nearly the same extraction efficiency (94.3%) and reproducibility (4.3% RSD) as the diluted sample (95.4%, 3.6% RSD). Figure 3 displays the efficiency under each condition.
Figure 3
 Discussion: ILE 96-Well Plates have exhibited the capacity to efficiently and reproducibly extract directly from serum samples without many of the preparative steps associated with other methods. The ILE method avoids sorbent conditioning, protein precipitation, sample dilution and solvent exchange/reconstitution. Further, ILE Well Plates do not clog, and the procedure completely eliminates the possibility of the formation of emulsions.
The simplicity of the ILE method allows an analyst to be confident with his data, as additional steps in any procedure invariably introduce greater possibility of error or imprecision. Immobilized Liquid Extraction 96-Well Plates offer a simplistic and elegant approach to the high-throughput extraction of small molecules directly from serum.
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