Application of 3D-printed scabbard-like sorbent for sample preparation in bioanalysis expanded to 96-wellplate high-throughput format.

Modern bioanalysis, which involves the quantitative and qualitative determination of small-molecule endogenous and exogenous substances in biological samples, is a powerful and useful tool that can generate valuable information related to many areas connected with human health and quality of life. Although LC-MS and GC-MS are widely viewed as the gold standards for many bioanalytical tasks, the scientific community has not abandoned its search for newer, more efficient, and more inexpensive methods of performing extraction as a sample preparation step before final analysis. Recent research showing the immense potential of 3D printing compelled our group to explore how this technology could be applied to techniques used in analytical chemistry. In particular, 3D printing offers three promising advantages: availability, low cost of materials and equipment, and the ability to fabricate objects of nearly any shape to suit the needs of a given application. Previously, we demonstrated that a commercial 3D material (LAY-FOMM) can function as a chemically active object that enables the reversible sorption of the antidiabetic drug, glimepiride, and endogenous steroids. In this report, we use a 3D printer to fabricate sorbents with a scabbard-like shape for use with a 96-blade system, which, along with the use of a 96-well plate, allows multiple extractions to be performed simultaneously. In order to assess the relative benefits of this 3D printed approach, we compare the performance of the proposed LAY-FOMM-based sorbent to that of the widely used C18 sorbent. Although the LAY-FOMM sorbent showed lower extraction recovery rates than the C18 sorbent, all of the other validation parameters suggest that it is suitable for use in high-throughput analysis of steroids in human plasma.

New 3D-printed sorbent for extraction of steroids from human plasma preceding LC-MS analysis.

In recent years, there has been an increasing worldwide interest in the use of alternative sample preparation methods that are proceeded by separation techniques. Fused deposition modeling (FDM) is a 3D printing technique that is based the consecutive layering of softened/melted thermoplastic materials. In this study, a group of natural steroids and sexual hormones - namely, aldosterone, cortisol, ß-estradiol, testosterone, dihydrotestosterone, and synthetic methyltestosterone and betamethasone - were separated and determined using an optimized high-performance liquid chromatography coupled to mass spectrometry (LC-MS) method in positive ionization mode. 3D-printed sorbents were selected as the pre-concentration technique because they are generally low cost, fast, and simple to make and automate. Furthermore, the use of 3D-printed sorbents helps to minimize potential errors due to their repeatability and reproducibility, and their ability to eliminate carry over by using one printed sorbent for a single extraction of steroids from biological matrices. The extraction procedure was optimized and the parameters influencing 3D-printed Layfomm 60® based sorbent and LC-MS were studied, including the type of extraction solvent used, sorption and desorption times, temperature, and the salting-out effect. To demonstrate this method's applicability for biological sample analysis, the SPME-LC-MS method was validated for its ability to simultaneously quantify endogenous steroids. This evaluation confirmed good linearity and an R2 that was between 0.9970 and 0.9990. The recovery rates for human plasma samples were 86.34-93.6% for the studied steroids with intra- and inter-day RSDs of 1.44-7.42% and 1.44-9.46%, respectively. To our knowledge, this study is the first time that 3D-printed sorbents have been used to extract trace amounts of endogenous low-molecular-weight compounds, such as steroids, from biological samples, such as plasma.

Bioanalysis of underivatized amino acids in non-invasive exhaled breath condensate samples using liquid chromatography coupled with tandem mass spectrometry.

Exhaled breath condensate (EBC) is receiving increased attention as a novel, entirely non-invasive technique for collecting biomarker samples. This increased attention is due to the fact that EBC is simple, effort independent, rapid, can be repeated frequently, and can be performed on young children and patients suffering from a variety of diseases. By having a subject breathe tidally through a cooling system for 15-20 min, a sufficient amount of condensate is collected for analysis of biomarkers in clinical studies. However, bioanalysis of EBC involves an unavoidable sample preparation step due to the low concentration of its components. Thus, there is a need for a new and more sensitive analytical method of assessing EBC samples. While researchers have considered analyses of single and small quantities of amino acids - for example, those connected with leukemia - no one has previously attempted to simultaneously analyze a panel of 23 amino acids. Moreover, the present study is well-justified, as prior studies focusing on single amino acids and leukemia at the moment of diagnosis and during chemotherapy (33 days of treatment) are inconsistent. In the present study, amino acids were separated using an XBridge Amide column (3 mm × 100 mm, 3.5 µm). The mobile phase consisted of 10 mM of ammonium buffer in water with a pH of 3 (Phase A) and 10 mM ammonium buffer in acetonitrile (Phase B) under gradient program elution. The analytes were detected in electrospray positive ionization mode. Under optimal conditions, the proposed method exhibited limits of quantification (LOQ) in the range of 0.05-0.5 ng/mL, and good linearity, with the determination coefficient (R2) falling between 0.9904 and 0.9998. The accuracy in human exhaled breath condensate samples ranged between 93.3-113.3% for the 23 studied amino acids, with intra- and inter-day coefficient of variation (CVs) of 0.13-9.92% and 0.17-10.53%, respectively. To demonstrate the liquid chromatography with hydrophilic interaction with electrospray source coupled to tandem mass spectrometry (LC-HILIC-ESI-MS/MS) method's applicability for biomedical investigations, it was verified and applied to determine amino acids in pediatric patients with leukemia. These tests confirmed that glutamine, arginine, homoarginine, asparagine, histidine, methionine, proline, hydroxyproline, threonine, tyrosine, and valine were present in significantly higher levels in pediatric leukemia patients than in the healthy control group. The developed assay is an attractive alternative to standard analytical methods, because it allows for the non-invasive, fast, sensitive, and reliable analysis of amino acids without derivatization in EBC.