March 1, 2014 (Vol. 34, No. 5)

The Advantages of Paper Spray MS over Conventional MS Include No Sample Prep

The need for rapid quantitative measurements on biological samples for drugs and metabolites is ever increasing as decisions in medicine become more reliant on personalized treatment. Medicine, we hope, in the 21st century will be less tied to averages and more on getting the right dose to the right patient at the right time. This inevitably increases the number of measurements necessary in clinical, toxicology, and analytical laboratories and supports a need for new methods that deliver faster time-to-results and with sufficient performance to address the application.

The analysis of blood and urine for illicit substances, therapeutic drugs, and other small molecules is commonly performed by an enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA), or liquid or gas chromatography combined with mass spectrometry. While liquid chromatography (LC) combined with mass spectrometry (MS) (i.e., LC-MS) is the gold standard for biochemical analysis, it suffers from a number of drawbacks that limit its application.

The most significant drawback of the LC-MS workflow is the time, skill, and equipment required for proper sample preparation. Furthermore, the complexity and cost of maintaining the LC is a barrier to many laboratories without highly skilled labor. As a result, new methods for direct sample analysis have been developed that aim to circumvent some of these challenges.

This tutorial will describe a recent method coined “Paper Spray” mass spectrometry, which overcomes the aforementioned limitations and offers several key advantages for rapid blood and urine analysis.

Paper Spray

Paper Spray is a simple method combining paper chromatography substrates and electrospray ionization. When combined with mass spectrometry, it’s a highly sensitive, selective, and versatile approach to screening and quantitating drugs in biological fluids. The analysis is simple and is performed by first depositing the sample directly onto a disposable cartridge. The sample is absorbed into the cellulose matrix, allowed to dry and inserted into the Paper Spray autosampler for introduction to the mass spectrometer.

The autosampler performs all of the steps necessary to perform the analysis prior to mass spectrometry detection including loading the cartridge, depositing the extraction solvent onto the cartridge, and positioning the cartridge in front of the mass spectrometer inlet. When solvent is deposited onto the cartridge, it migrates through the sample by capillary action, dissolving and eluting the analytes of interest. A high voltage is applied to the cartridge to produce an electrospray of the sample. Figure 1 depicts a Paper Spray autosampler (Velox™ Paper Spray System) developed by Prosolia.

Figure 1. Computer-generated model of the Velox Paper Spray System developed by Prosolia


Paper Spray delivers the advantages of speed, quantitative capacity, use of low volumes of sample (i.e., 10–20 µLs), and ease of use. Utilizing the inherent advantages of the mass spectrometer in selectivity and sensitivity, Paper Spray MS yields a powerful method for rapid screening and quantitation for drugs in biological matrices such as urine, plasma, and even whole blood.

Quantitation by Paper Spray is achieved by spiking a known quantity of a stable isotope-labeled analog of the analyte into the sample prior to analysis, much the same as with traditional HPLC-MS assays. Unlike with HPLC, however, the sample is then analyzed directly and without chemical separations prior to MS.

Table compares the various performance attributes of Paper Spray-MS/MS and LC-MS/MS for the quantitation of imatinib, a cancer treatment drug, in whole blood. Significant time savings is achieved when using Paper Spray, while both methods yield the same analytical performance.

Comparison of Method Characteristics

Paper Spray has been used in academic, pharmaceutical, and clinical research laboratories for quantitative analysis of drugs and drug metabolites. Some validated drug assays developed by Paper Spray include chemotherapeutics such as melphalan, the selective estrogen receptor modulator tamoxifen, targeted cancer therapeutics such as imatinib and pazopanib, and immunosuppressive drugs such as tacrolimus.

Assay validation typically includes establishing that the method meets preset criteria for accuracy and precision, and that the analysis method has adequate selectivity in the face of similar drug or drug metabolites that could interfere. For each of the drugs listed, samples were also split and run on a traditional HPLC-MS or MS/MS platform to further validate the accuracy of Paper Spray; in each case the concentrations determined by Paper Spray MS and HPLC-MS were comparable.

Figure 2. (A) Paper Spray mass spectrum of a urine sample obtained anonymously. The data was recorded using a Thermo Scientific Exactive™ mass spectrometer.

Figure 2A shows a Paper Spray mass spectrum of a urine sample obtained anonymously from a toxicology laboratory revealing high levels of methamphetamine, tramadol (or desmethyl-venlafaxine), EDDP (2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine), and methadone. Peak assignments were made by exact mass measurements using the Thermo Scientific Exactive™ high-resolution mass spectrometer.

Figure 2B shows the selected ion chronogram for increasing concentrations of EDDP spiked into synthetic urine. Peak widths in the paper spray chronograms are a function of the time with which the high voltage is applied to the cartridge; in this case it was 60 seconds for each sample, although shorter analysis times are possible.

Figure 2. (B) Selected ion chronogram of increasing concentrations of EDDP in urine.


With the development of Paper Spray ionization and the Velox, a new bioanalytical tool is presented having the advantages of no sample preparation, requiring only a few microliters of sample, and speed compared to conventional LC-MS. The methodology has compelling advantages and presents unique possibilities in forensic toxicology and drug development. While the present state of the technology is not as sensitive as state-of-the-art LC-MS, for applications requiring modest limits of quantitation (e.g., detection of illicit drugs in blood, urine, and other biofluids) it is well suited.

Justin M. Wiseman ([email protected]) is chairman, president, and CEO of Prosolia, and Nicholas E. Manicke is assistant professor of chemistry at IUPUI.

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