Recent advances in sampling techniques in the pharmaceutical industry sparked significant interest in applying improvements
to extraction methods for greater analyte detection and quantitation. In particular, the dried blood spot (DBS) sampling technique
has numerous advantages compared to traditional methods such as liquid–liquid extraction, including the use of small sample
volumes, less sample processing, and less exposure to toxic solvents (ether, methyl tert-butyl ether [MTBE], and dichloromethane). In this article, we discuss the adaptation of DBS technology to develop and validate
a novel paper strip extraction method for the analysis of natural product metabolites in biological samples obtained from
a human pharmacokinetic study of xanthohumol, a hop prenylflavonoid.
Globally, chronic diseases serve as the leading cause of death (1,2) and are projected to be responsible for 64 million deaths
annually by 2015 (1). The economic and social impact of chronic diseases necessitated continuous advances in analytical techniques
for the development and discovery of therapeutic agents. Improvements in mass spectrometry (MS) technology have allowed for
the creation of high-throughput methods, which are used in quantitative analysis of drugs and metabolites in biological samples.
The complexity of biological samples (that is, plasma, urine, feces, and so on) influences the sensitivity and selectivity
of MS methods and emphasizes the importance of sample preparation. Proteins, salts, and organic compounds present in biological
samples may interfere with the detection of the analytes of interest, particularly if they are present in trace amounts, thus
requiring protocols for extraction, isolation, or concentration of analytes from samples, which may affect accuracy and reproducibility
of analyte measurements (3–9).
Common sample preparation methods include liquid–liquid extraction (LLE) and solid-phase extraction (SPE). Traditionally,
LLE has been the prominent extraction method for the analysis of natural products (10) and one of its main strengths is providing
aqueous and organic fractions during analysis. However, two major limitations of LLE (sample preparation time and toxic solvent
use) (10) have led to the emergence of SPE as a preferred extraction method. SPE lowers analysis costs, organic solvent use,
and time compared to LLE (10). SPE involves the adsorption of analytes on solid material before elution with the solvent and
has been used in clinical toxicological analysis of drugs (11). Disadvantages of SPE methodology include sample volume and
the potential need for optimization of SPE methods including adsorptive material and elution solvent for analytes. Challenges
with SPE methods prompted a search for alternative sample preparation methods, specifically dried blood spot (DBS) sampling.
Interest in DBS sampling has grown significantly, particularly within the pharmaceutical industry because of the lower costs
associated with the technique. The DBS technique consists of collecting and drying a biological sample on a small filter paper
disk and processing for subsequent liquid chromatography–tandem mass spectrometry (LC–MS-MS) analysis. With the use of filter
paper, DBS uses the adsorptive properties of cellulose for analyte extraction, which may lead to lower costs compared to the
adsorptive material of SPE (for example, C18). The cellulose fibers in the filter paper aid in the removal of matrix components
that interfere with analyte detection (that is, protein precipitation from plasma samples). Thomas and colleagues (12) illustrated
the use of DBS in the detection of various drugs and their phase I and II metabolites. DBS has numerous advantages including
minimization of analyte loss, the use of small sample volumes, fewer sample processing steps, low matrix effects, and less
exposure to toxic organic solvents compared to traditional methods such as LLE (13).
All of the current sample extraction methods (DBS, SPE, and LLE) have advantages and limitations. The aim of this study is
to combine aspects of DBS, SPE, and LLE methods to develop and validate a novel paper strip extraction (PSE) method that maximizes
the strength of each while eliminating the weaknesses. The PSE method uses an LC–MS-MS compatible elution solvent for analyte
extraction. PSE also incorporates filter paper as its adsorption material, similar to DBS, but enlarges size and alters shape
to account for larger sample size and eliminates one of the drawbacks of DBS — the need for high analyte concentration because
of small samples, which dictates a small concentration range for analyte detection. We evaluated the PSE method in the analysis
of natural product metabolites (Figure 1) in biological samples obtained from a human pharmacokinetic study of xanthohumol,
a hop prenylflavonoid.
Figure 1: Structures of analytes of interest: natural product metabolites of xanthohumol, a hop prenylflavonoid with antiobesity