A microfluidic technique for quantification of steroids in core needle biopsies.

Core needle biopsy (CNB) sampling is known to be inexpensive and minimally invasive relative to traditional tissue resectioning. But CNBs are often not used in analytical settings because of the tiny amount of sample and analyte. To address this challenge, we introduce an analytical method capable of multiplexed steroid quantification in CNB samples-those studied here ranged in mass from 2 to 8 mg. The new method uses digital microfluidics to extract steroids from CNB tissue samples (including a solid-phase extraction cleanup step) followed by analysis by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). The method has limits of detection of 3.6, 1.6, 5.8, and 8.5 fmol for estradiol, androstendione, testoterone, and progesterone, respectively. We propose that future generations of this method may be useful for regular quantification of steroids in core needle biopsy samples of breast tissue to inform dosage and timing of antihormone or hormone replacement therapies as part of a personalized medicine approach to treating a variety of hormone-sensitive disorders.

Attractive design: an elution solvent optimization platform for magnetic-bead-based fractionation using digital microfluidics and design of experiments.

There is great interest in the development of integrated tools allowing for miniaturized sample processing, including solid phase extraction (SPE). We introduce a new format for microfluidic SPE relying on C18-functionalized magnetic beads that can be manipulated in droplets in a digital microfluidic platform. This format provides the opportunity to tune the amount (and potentially the type) of stationary phase on-the-fly, and allows the removal of beads after the extraction (to enable other operations in same device-space), maintaining device reconfigurability. Using the new method, we employed a design of experiments (DOE) operation to enable automated on-chip optimization of elution solvent composition for reversed phase SPE of a model system. Further, conditions were selected to enable on-chip fractionation of multiple analytes. Finally, the method was demonstrated to be useful for online cleanup of extracts from dried blood spot (DBS) samples. We anticipate this combination of features will prove useful for separating a wide range of analytes, from small molecules to peptides, from complex matrices.

Analysis on the go: quantitation of drugs of abuse in dried urine with digital microfluidics and miniature mass spectrometry.

We report the development of a method coupling microfluidics and a miniature mass spectrometer, applied to quantitation of drugs of abuse in urine. A custom digital microfluidic system was designed to deliver droplets of solvent to dried urine samples and then transport extracted analytes to an array of nanoelectrospray emitters for analysis. Tandem mass spectrometry (MS/MS) detection was performed using a fully autonomous 25 kg instrument. Using the new method, cocaine, benzoylecgonine, and codeine can be quantified from four samples in less than 15 min from (dried) sample to analysis. The figures of merit for the new method suggest that it is suitable for on-site screening; for example, the limit of quantitation (LOQ) for cocaine is 40 ng/mL, which is compatible with the performance criteria for laboratory analyses established by the United Nations Office on Drugs and Crime. More importantly, the LOQ of the new method is superior to the 300 ng/mL cutoff values used by the only other portable analysis systems we are aware of (relying on immunoassays). This work serves as a proof-of-concept for integration of microfluidics with miniature mass spectrometry. The system is attractive for the quantitation of drugs of abuse from urine and, more generally, may be useful for a wide range of applications that would benefit from portable, quantitative, on-site analysis.

Multiplexed extraction and quantitative analysis of pharmaceuticals from DBS samples using digital microfluidics.

BACKGROUND: Dried blood spot (DBS) sampling is emerging as a valuable technique in a variety of fields, including clinical and preclinical testing of pharmaceuticals. Despite this popularity, current DBS sampling and analysis processes remain laborious and time consuming. Digital microfluidics, a microscale liquid-handling technique, characterized by the manipulation of discrete droplets on open electrode arrays, offers a potential solution to these problems. RESULTS: We report a new digital microfluidic method for multiplexed extraction and analysis of pharmaceuticals in DBS samples. In the new method, four DBS samples are extracted in microliter-sized droplets containing internal standard, and the extract is delivered to dedicated nanoelectrospray ionization emitters for direct analysis by tandem mass spectometry and selected reaction monitoring. CONCLUSION: The new method allows for an order of magnitude reduction in processing time and approximately three-times reduction in extraction solvent relative to conventional techniques, while maintaining acceptable analytical performance for most drugs tested.

A digital microfluidic method for dried blood spot analysis.

Blood samples stored as dried blood spots (DBSs) are emerging as a useful sampling and storage vehicle for a wide range of applications. Unfortunately, the surging popularity of DBS samples has not yet been accompanied by an improvement in automated techniques for extraction and analysis. As a first step towards overcoming this challenge, we have developed a prototype microfluidic system for quantification of amino acids in dried blood spots, in which analytes are extracted, mixed with internal standards, derivatized, and reconstituted for analysis by (off-line and in-line) tandem mass spectrometry. The new method is fast, robust, precise, and most importantly, compatible with automation. We propose that the new method can potentially contribute to a new generation of analytical techniques for quantifying analytes in DBS samples for a wide range of applications.

Detection of acute fentanyl exposure in fresh and decomposed skeletal tissues part II: the effect of dose-death interval.

The effects of dose-death interval on the detection of acute fentanyl exposure in fresh and decomposed skeletal tissues (marrow and bone), by automated enzyme-linked immunosorbent assay (ELISA) are described. Rats (n=14) were administered fentanyl acutely at a dose of 0 (n=2) or 60 microg/kg (n=12) by intraperitoneal injection, and euthanized within 20, 45, 135, or 225 min. Femora and tibiae were extracted from the fresh corpses and marrow was isolated from the femoral and tibial medullary cavities. The remains were then allowed to decompose outdoors to the point of complete skeletonization, and vertebrae, pelvi and miscellaneous (humeri and scapulae) were recovered for analysis. In all cases, bones were cleaned in alkaline solution and then ground into a fine powder. Marrow was homogenized in alkaline solution. Fentanyl was extracted from ground bone by methanolic extraction. Extracts were adjusted to pH 6 and analyzed by ELISA. Perimortem heart blood was also collected and diluted in phosphate buffer prior to screening by ELISA. The effect of tissue type on ELISA response was examined through determination of binary classification test sensitivity and the relative decrease in absorbance (%DA, drug-positive tissues vs. drug-free controls) in each tissue type. Overall, the %DA varied significantly between extracts from different skeletal tissues at a given dose-death interval, according to the general order of marrow>decomposed bone>fresh bone. Binary classification test sensitivity values for fentanyl in marrow, fresh epiphyseal (femoral and tibial) bone, fresh diaphyseal (femoral and tibial) bone, decomposed vertebrae, decomposed pelvic bone, and decomposed miscellaneous bone were 67-100%, 0-33%, 0-33%, 0-67%, 0-67% and 0-33%, respectively, over all dose-death intervals. Although group mean %DA values showed a strong negative correlation with dose-death interval in marrow, fresh epiphyseal bone, decomposed vertebrae, pelvic and miscellaneous bone (r=-0.989, -0.930, -0.955, -0.903, and -0.974, respectively), the high variability in both fresh and decomposed bone precluded differentiation of the dose-death intervals based on %DA value alone. Overall, the results suggested that the type of skeletal tissue sampled may not be as important as the amount of residual marrow remaining in skeletonized remains.

Detection of acute fentanyl exposure in fresh and decomposed skeletal tissues.

The detection of acute fentanyl exposure in fresh and decomposed skeletal tissues (marrow and bone), by automated enzyme-linked immunosorbent assay (ELISA) is described. Rats (n=15) were administered fentanyl acutely at a dose of 0 (n=3), 15 (n=3), 30 (n=3) or 60 microg/kg (n=6) by intraperitoneal injection, and euthanized within 20 min. Femora and tibiae were extracted from the fresh corpses and marrow was isolated from the femoral and tibial medullary cavities. The remains were then allowed to decompose outdoors to the point of complete skeletonization, and vertebrae and pelvi were recovered for analysis. In all cases, bones were cleaned in alkaline solution and then ground into a fine powder. Marrow was homogenized in alkaline solution. Fentanyl was extracted from ground bone by methanolic extraction. Extracts were adjusted to pH 6 and analyzed by ELISA. Perimortem heart blood was also collected and diluted in phosphate buffer prior to screening by ELISA. The effect of tissue type on ELISA response was examined through determination of binary classification test sensitivity and the relative decrease in absorbance (%DA, drug-positive tissues vs drug-free controls) in each tissue type. Overall, the %DA varied significantly between extracts from different skeletal tissues under a given dose condition, according to the general order of marrow>vertebrae approximately pelvi>epiphyseal bone approximately diaphyseal bone. Binary classification test sensitivity values for fentanyl in marrow, fresh epiphyseal (femoral and tibial) bone, fresh diaphyseal (femoral and tibial) bone, decomposed vertebrae and decomposed pelvic bone were 100%, 16-33%, 0-16%, 0-33% and 66-100%, respectively, at the 60 microg/kg dose level. While mean %DA values showed a strong positive correlation with those in marrow and blood measurements and the administered dose (r=0.997 and 0.986), such a correlation was not observed in assays of decomposed tissues (r=-0.157 and -0.315). These results suggest that the type of skeletal tissue sampled and position within a given bone may be important considerations in the choice of substrate for fentanyl screening in skeletal tissues, but that quantitative analysis of drugs in decomposed bones may be of limited interpretive value.