Chemistry


by Carolin Gallert, Ellen Vorberg, Thomas Roddelkopf, Steffen Junginger, Heidi Fleischer and Kerstin Thurow


Evaluation of an Automated Solid-Phase Extraction Method Using Positive Pressure


I


nvestigations for the determination of drugs in real samples, while gaining in im- portance, are limited by time and sample throughput. In response to this, the authors developed an automated solid-phase extrac- tion (SPE) procedure using a liquid-handling platform with a positive pressure unit. This automated SPE was validated after the manual SPE process. HPLC/MS was used to determine drug concentration. The results showed that 1) automation offers a precise and robust sys- tem for sample preparation and 2) automated SPE is suitable for routine analysis with less manual processing and fewer operator errors.


Introduction Each year, 1.7 million people in the U.S. are


diagnosed with cancer; of these, approximately 580,000 will die from the disease.1,2


Demand for


pharmacological investigations, specifically the determination of drugs in real samples, has be- come increasingly important to optimize cancer treatment.3


Currently available automated SPE meth- ods have limitations or do not provide high throughput. Allanson et al.4


and Marinova et al.5


presented a highly precise automated SPE pro- cess performed on a liquid handling platform that used a vacuum module for sample process- ing. However, automated SPE systems working with vacuum have a number of disadvantages, including a maximum pressure difference of 1 bar on the columns and imbalanced, or ir- regular, sample processing. In the latter case, various SPE columns are emptied at different speeds. This is problematic because depleted columns can cause a reduction in vacuum, lead- ing to longer sample processing times due to a slower sample flow rate. It is important that the processing time for all columns is equal to ensure consistent sample handling and reli- able results.6


For complete sample processing,


vacuum is often applied to the columns for a longer period of time, even if they are already


empty; however, this extended drying time may have a negative impact on the SPE.


The positive pressure unit used in this study overcomes these drawbacks because it is able to operate at a higher pressure, which ranges from 0.1 to 7 bar and guarantees constant pressure on all columns. This results in defined and steady pressure being applied over a specified time. Moreover, the positive pressure unit can be con- trolled by liquid handlers to achieve automated SPE. The system also provides conditioning and rinsing of the columns.6


Manual and automated


SPE procedures Manual and automated SPE were compared for the quantification of the cytotoxic drug cyclophosphamide in biological samples. Cyclophosphamide is used in chemotherapy and in the treatment of scleroderma lung disease and multiple sclerosis.7–9


Manual SPE


with vacuum was used to extract cyclophos- phamide. The automated system consisted of a Biomek NX liquid handler (Beckman Coulter, Brea, Calif.) with a Span-8 pipetting head and positive pressure unit for sample processing (amplius GmbH, Rostock, Germany). The system provides precise and robust SPE, is adaptable to different methods, requires less manual labor and reduces errors compared to manual pro- cessing errors.


Materials and methods


Manual solid-phase extraction A Chromabond vacuum chamber (Macherey- Nagel, Düren, Germany) and Strata C18-E cartridges (Phenomenex,


Torrance, Calif.)


were used for manual liquid handling. Cyclophosphamide (Sigma-Aldrich, St. Louis, Mo.) was dissolved in Dulbecco’s Modified Eagle’s medium (DMEM) supplied with fetal calf serum (FCS), penicillin/streptomycin and glutamin (Sigma-Aldrich). To extract cyclo- phosphamide using the SPE process, Strata


AMERICAN LABORATORY • 28 • MARCH 2015


C18-E cartridges were conditioned with 2 mL of gradient-grade methanol ROTISOLV HPLC (Roth, Karlsruhe, Germany). The conditioning step was followed by preequilibration with 2 mL of Milli-Q organic-free water (Millipore, Bedford, Mass.). Subsequently, 1 mL of cyclo- phosphamide dissolved in DMEM was added and passed slowly through the column. Cartridges were washed with 2 mL of Milli-Q organic-free water and dried for 10 minutes. Finally, cyclophosphamide was eluted twice with 500 µL gradient-grade methanol ROTISOLV HPLC and samples were diluted accordingly.


Automated solid-phase extraction The system was validated with two different plates: 24 columns, which were stacked in an adapter plate, and a 96-deep-well collection plate (Phenomenex). These two plates were placed into the positive pressure unit (Figure 1). Positive pressure forced the liquids through the cartridges. The automated SPE steps were done in accordance with the manual procedure. An appropriate sample dilution was included in the automated process.


HPLC/MS An Agilent 1200 series system (Agilent Technologies, Santa Clara, Calif.) equipped with a LC/MS TOF, high-performance au- tosampler, binary pump and thermostated column compartment was used for the chro- matographic separation. Specific parameters of the HPLC/MS method for cyclophospha- mide determination are shown in Table 1. Cyclophosphamide detection was done with an Agilent autosampler SL, LC/MS TOF and electrospray ionization (ESI) interface. The retention time of cyclophosphamide was 3.59 minutes and 2.36 minutes for caffeine as the internal standard. In the positive ion mode, the molar mass was 261.03 g/mol for cyclo- phosphamide and 195.09 g/mol for caffeine. Qualitative and quantitative analyses were performed with Agilent MassHunter worksta- tion software.


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