Analysis Of Biological Samples Research Articles

Development and Validation of an LC-MS/MS Method for the Determination of Plasma and Red Blood Cell Omega Fatty Acids: A Useful Diagnostic Tool

Development and Validation of an LC-MS/MS Method for the Determination of Plasma and Red Blood Cell Omega Fatty Acids: A Useful Diagnostic Tool

Incorporation of Three Different Optical Trains into the IR-MALDESI Mass Spectrometry Imaging Platform to Characterize Artemisia annua.

Artemisinin is the leading medication for the treatment of malaria and is only produced naturally in Artemisia annua. The localization of artemisinin in both the glandular and non-glandular trichomes of the plant makes it an ideal candidate for mass spectrometry imaging (MSI) as a model system for method development. Infrared matrix-assisted laser desorption electrospray ionization MSI (IR-MALDESI-MSI) has the capability to detect hundreds to thousands of analytes simultaneously, providing abundance information in conjunction with species localization throughout a sample. The development of several new optical trains and their application to the IR-MALDESI-MSI platform has improved data quality in previous proof-of-concept experiments but has not yet been applied to analysis of native biological samples, especially the MSI analysis of plants. This study aimed to develop a workflow and optimize MSI parameters, specifically the laser optical train, for the analysis of Artemisia annua with the NextGen IR-MALDESI platform coupled to an Orbitrap Exploris 240 mass spectrometer. Two laser optics were compared to the conventional set up, of which include a Schwarzschild-like reflective objective and a diffractive optical element (DOE). These optics, respectively, enhance the spatial resolution of imaging experiments or create a square spot shape for top-hat imaging. Ultimately, we incorporated and characterized three different optical trains into our analysis of Artemisia annua to study metabolites in the artemisinin pathway. These improvements in our workflow, resulted in high spatial resolution and improved ion abundance from previous work, which will allow us to address many different questions in plant biology beyond this model system.

Biomonitoring of volatile organic compounds and organophosphorus flame retardands in commercial aircrews after „fume and smell events“

Health risks to humans after „fume and smell events“, short-term incidents on aircrafts that are accompanied by unpleasant odour or visible smoke, remain a subject of controversy. We assessed exposure to volatile organic compounds (VOC) and organophosphorus compounds (OPC) by biomonitoring in 375 aircrew members after self-reported “fume and smell events” and in 88 persons of the general population. A total of 20 parameters were analysed in blood and urine by gas chromatography and mass spectrometry. Median levels of acetone in blood and urine and 2-propanol in blood were elevated in aircrews compared to controls (p < 0.0001). Additionally, elevated peak exposures, best estimated by the 95th percentiles, were observed in aircrews for n-heptane and n-octane in blood, and acetone, 2,5-hexanedione and o-cresol in urine. Only the maximum observed levels of 2,5-hexandione in urine (768 μg/L) and toluene in blood (77 μg/L) in aircrew members were higher than the current biological exposure indices (BEI® levels) (500 and 20 μg/L, respectively) of the American Conference of Governmental Industrial Hygienists (US-ACGIH) for workers occupationally exposed to n-hexane and toluene, two well-accepted human neurotoxicants. Low-level exposures to n-hexane and toluene could be also observed in controls. The majority of OPC parameters in urine, including those of neurotoxic ortho-isomers of tricresylphosphate, were below the limit of quantitation in both aircrews and controls. Our comparative VOC and OPC analyses in biological samples of a large number of aircrew members and controls suggest that exposures are similar in both groups and generally low.

Development of CO2-switchable deep eutectic solvent-based liquid-liquid microextraction approach: Application in urinary excretion and tissue distribution studies

BackgroundPharmacokinetic studies are pivotal in drug development, focusing on absorption, distribution, and excretion of active compounds. Effective sample preparation methods play a crucial role in these studies. Traditional techniques like protein precipitation and liquid-liquid extraction often involve toxic solvents and are time-consuming. Recently, deep eutectic solvent (DES) has emerged as an eco-friendly alternative due to its high efficiency, low cost, and low toxicity. This study introduces a novel sample pretreatment method using CO2-switchable DES in liquid-liquid microextraction (LLME) to enhance speed, accuracy, and sensitivity in complex biological samples analysis. ResultsA liquid-liquid microextraction sample pretreatment method based on switchable DES combined with high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was established for the analysis of urine and tissue samples. The method was optimized through systematic investigation of key parameters, including DES type, volume, molar ratio, pH, vortex time, gas purge time, and salt addition. The resulting procedure exhibited satisfying linearity (r2 ≥ 0.9958), good precision (RSD ≤6.01 %), desirable recovery (52.44%–98.12 %) and matrix effect (86.22%–119.30 %), and the accuracy and precision of stability were within the ±15 % limit. The proven methods were further applied to urinary excretion study and tissue distribution study of Nelumbinis plumula (NP) extract. The results indicated that the total cumulative excretion of liensinine, isoliensinine and neferine in urine within 240 h was 4.96 %, 0.66 % and 0.44 %, respectively. The tissue distribution study showed that alkaloids mainly distribute in liver, kidney, and spleen. SignificanceThis research introduces a groundbreaking technique distinguished by its simplicity, speed, cost-effectiveness, and environmental friendliness. This approach, utilizing CO2-switchable DES as an extraction solvent for LLME, integrates deproteinization and removal of interfering molecules into a single step. This integration showcases its efficiency and convenience, demonstrating significant promise for various applications in the analysis of biological samples. Additionally, this study provides the first report on urinary excretion and tissue distribution of alkaloids from NP using a DES-LLME method. These findings offer valuable insights into the in vivo behavior of herbal medicine, enhancing understanding of pharmacological actions and facilitating clinical rational administration.

Bridging biological samples to functional nucleic acid biosensor applications: current enzymatic-based strategies for single-stranded DNA generation.

The escalating threat of emerging diseases, often stemming from contaminants and lethal pathogens, has precipitated a heightened demand for sophisticated diagnostic tools. Within this landscape, the functional nucleic acid (FNA) biosensor, harnessing the power of single-stranded DNA (ssDNA), has emerged as a preeminent choice for target analyte detection. However, the dependence on ssDNA has raised difficulties in realizing it in biological samples. Therefore, the production of high-quality ssDNA from biological samples is critical. This review aims to discuss strategies for generating ssDNA from biological samples for integration into biosensors. Several innovative strategies for ssDNA generation have been deployed, encompassing techniques, such as asymmetric PCR, Exonuclease-PCR, isothermal amplification, biotin-streptavidin PCR, transcription-reverse transcription, ssDNA overhang generation, and urea denaturation PAGE. These approaches have been seamlessly integrated with biosensors for biological sample analysis, ushering in a new era of disease detection and monitoring. This amalgamation of ssDNA generation techniques with biosensing applications holds significant promise, not only in improving the speed and accuracy of diagnostic processes but also in fortifying the global response to deadly diseases, thereby underlining the pivotal role of cutting-edge biotechnology in public health and disease prevention.

Preparation of a β-cyclodextrin grafted magnetic biochar for efficient extraction of four antiepileptic drugs in plasma samples

Simultaneous monitoring of plasma concentration levels of multiple antiepileptic drugs (AEDs) is essential for dose adjustment in comprehensive epilepsy treatment, necessitating a sensitive technique for accurate extraction and determination of AEDs. Herein, a magnetic solid-phase extraction (MSPE) technique on the basis of modified biochar (BC) is investigated to extract four AEDs from plasma, in conjunction with high performance liquid chromatography. BC derived from Zizyphus jujuba seed shells was activated by phosphoric acid (PBC) and magnetized via coprecipitation to produce MPBC. The MPBCCD obtained after modification with β-cyclodextrin (CD) was characterized and evaluated for adsorption. It exhibited fast adsorption kinetics based on second-order kinetics and satisfactory adsorption capacity for AEDs. Then it was employed as the MSPE adsorbent and the influencing parameters were optimized. The enrichment factor was 18.75. The validation analysis revealed a favorable linearity that ranged from 0.04 to 20 μg·mL−1 along with a low limit of detection of 6.85 to 10.19 ng·mL−1. The recovery of the AEDs ranged from 78.7 to 109.2 %, with relative standard deviations below 6.7 %. Using quantum chemistry theory calculations and experimental results analysis, the adsorption mechanism was investigated. It disclosed that the suggested strategy built upon MPBCCD was appropriate for the assessment of AEDs in plasma and expanded the usage of BC as the environmentally favorable matrix for the analysis of biological samples.

Determination of Δ9-tetrahydrocannabinol, 11-nor-carboxy-Δ9-tetrahydrocannabinol and cannabidiol in human plasma and urine after a commercial cannabidiol oil product intake

PurposeCannabidiol (CBD) products are widely used for pain relief, sleep improvement, management of seizures etc. Although the concentrations of Δ9-tetrahydrocannabinol (Δ9-THC) in these products are low (≤0.3% w/w), it is important to investigate if its presence and/or that of its metabolite 11-nor-carboxy-Δ9-THC, is traceable in plasma and urine samples of individuals who take CBD oil products.MethodsA sensitive GC/MS method for the determination of Δ9-THC, 11-nor-carboxy-Δ9-THC and CBD in plasma and urine samples was developed and validated. The sample preparation procedure included protein precipitation for plasma samples and hydrolysis for urine samples, solid-phase extraction and finally derivatization with N,O-bis(trimethylsilyl)trifluoroacetamide) with 1% trimethylchlorosilane.ResultsFor all analytes, the LOD and LOQ were 0.06 and 0.20 ng/mL, respectively. The calibration curves were linear (R2 ≥ 0.992), and absolute recoveries were ≥91.7%. Accuracy and precision were within the accepted range. From the analysis of biologic samples of 10 human participants who were taking CBD oil, it was realized that Δ9-THC was not detected in urine, while 11-nor-carboxy-Δ9-THC (0.69–23.06 ng/mL) and CBD (0.29–96.78 ng/mL) were found in all urine samples. Regarding plasma samples, Δ9-THC (0.21–0.62 ng/mL) was detected in 10, 11-nor-carboxy-Δ9-THC (0.20–2.44 ng/mL) in 35, while CBD (0.20–1.58 ng/mL) in 25 out of 38 samples, respectively.ConclusionThe results showed that Δ9-THC is likely to be found in plasma although at low concentrations. In addition, the detection of 11-nor-carboxy-Δ9-THC in both urine and plasma samples raises questions and concerns for the proper interpretation of toxicological results, especially considering Greece’s zero tolerance law applied in DUID and workplace cases.

Abstract CT139: Immunological activity of oncolytic adenovirus encoding TNFα and IL-2 (TILT-123) in combination with pembrolizumab in platinum resistant or refractory ovarian cancer patients

Abstract Background TILT-123 (Ad5/3-E2F-D24-hTNFα-IRES-hIL-2) is a serotype chimeric dual safety device oncolytic adenovirus armed with tumor necrosis factor alpha and interleukin-2. It was designed to improve efficacy of T-cell therapies such as immune checkpoint blockade or adoptive cell transfer. We present biological and immunological endpoints from an open-label phase 1 dose escalation study of TILT-123 in combination with anti-PD-1 pembrolizumab in platinum resistant or refractory ovarian cancer patients. Experimental procedures In this dose escalation trial (NCT05271318), patients were first injected with an intravenous (i.v.) dose of TILT-123 ranging from 3x1011 to 4x1012 virus particles (VP), followed by five intratumoural (i.t.) or intraperitoneal (i.p.) injections ranging from 1x1011 to 5x1011 VP. Patients also received intravenous injections of 200 mg pembrolizumab every three weeks starting 36 days after the first dose of TILT-123. The primary endpoint was safety, while efficacy by RECIST1.1 was a secondary endpoint. Collection of tumor biopsies, circulating immune cells, serum, saliva, urine and feces enabled multi-omic evaluation of mechanism of action and identification of predictive biomarkers. Summary of data As of January 2nd 2024, 15 patients received trial therapy. No dose-limiting toxicities were observed and the most frequent AEs associated with therapy included chills and nausea (&amp;gt;10% of patients). Stable disease by RECIST 1.1 was seen in 67% in 12 evaluable patients. TILT-123 replication was detected in both injected and non-injected tumors as assessed by qPCR and anti-hexon IHC. Multiplex immunofluorescence demonstrated induction of robust immunological activity in both injected and non-injected tumors, including significant increase in granzyme B+, PD-1+ CD4+ and CD8+ T cells as well as PD-1+ NK cells. Baseline intratumoral CD4+, FOXP3+ regulatory T cells were not associated with clinical response. Tertiary lymphoid structures and lymphoid aggregates were identified in patients showing long term disease control. Low to medium titers of neutralizing antibodies against 5/3 chimeric adenovirus were detected in 57% of patients at baseline and were not a predictive factor of disease control. No patients had high titers at baseline. Conclusion TILT-123 in combination with anti-PD-1 pembrolizumab was safe and resulted in disease control in both platinum resistant and refractory ovarian cancer patients. Analysis of biological samples revealed insights into mechanism of action, including an immune profile predictive of clinical response. These results, together with those from four other trials (NCT04695327, NCT04217473, NCT06125197 and NCT05222932) set the stage for continued clinical evaluation of TILT-123. Citation Format: James H. Clubb, Matthew S. Block, Johanna Mäenpää, Santeri Pakola, Lyna Haybout, Dafne C. Quixabeira, Tatiana Kudling, Mirte van der Heijden, Elise Jirovec, Tuomo Alanko, Daniel A. Adamo, Susan Ramadan, Jorma Sormunen, Juha Kononen, Julia W. Cohen, Nadena Singh, John Goldfinch, Suvi Sorsa, Riikka Havunen, Claudia Kistler, João M. Santos, Víctor Cervera-Carrascon, Akseli Hemminki. Immunological activity of oncolytic adenovirus encoding TNFα and IL-2 (TILT-123) in combination with pembrolizumab in platinum resistant or refractory ovarian cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT139.

Enhancing comparative T cell receptor repertoire analysis in small biological samples through pooling homologous cell samples from multiple mice

Accurate characterization and comparison of Tcell receptor (TCR) repertoires from small biological samples present significant challenges. The main challenge is the low material input, which compromises the quality of bulk sequencing and hinders the recovery of sufficient TCR sequences for robust analyses. We aimed to address this limitation by implementing a strategic approach to pool homologous biological samples. Our findings demonstrate that such pooling indeed enhances the TCR repertoire coverage, particularly for cell subsets of constrained sizes, and enables accurate comparisons of TCR repertoires at different levels of complexity across Tcell subsets with different sizes. This methodology holds promise for advancing our understanding of Tcell repertoires in scenarios where sample size constraints are a prevailing concern.

Development of an LC-MS/MS method for the determination of five psychoactive drugs in postmortem urine by optimization of enzymatic hydrolysis of glucuronide conjugates.

Toxicological analyses of biological samples play important roles in forensic and clinical investigations. Ingested drugs are excreted in urine as conjugates with endogenous substances such as glucuronic acid; hydrolyzing these conjugates improves the determination of target drugs by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In this study, we sought to improve the enzymatic hydrolysis of glucuronide conjugates of five psychoactive drugs (11-nor-9-carboxy-Δ9-tetrahydrocannabinol, oxazepam, lorazepam, temazepam, and amitriptyline). The efficiency of enzymatic hydrolysis of glucuronide conjugates in urine was optimized by varying temperature, enzyme volume, and reaction time. The hydrolysis was performed directly on extraction columns. This analysis method using LC-MS/MS was applied to forensic autopsy samples after thorough validation. We found that the recombinant β-glucuronidase B-One® quantitatively hydrolyzed these conjugates within 3min at room temperature directly on extraction columns. This on-column method saved time and eliminated the loss of valuable samples during transfer to the extraction column. LC-MS/MS-based calibration curves processed with this method showed good linearity, with r2 values exceeding 0.998. The intra- and inter-day accuracies and precisions of the method were 93.0-109.7% and 0.8-8.8%, respectively. The recovery efficiencies were in the range of 56.1-104.5%. Matrix effects were between 78.9 and 126.9%. We have established an LC-MS/MS method for five psychoactive drugs in urine after enzymatic hydrolysis of glucuronide conjugates directly on extraction columns. The method was successfully applied to forensic autopsy samples. The established method will have broad applications, including forensic and clinical toxicological investigations.

Dual toeholds regulated CRISPR-Cas12a sensing platform for ApoE single nucleotide polymorphisms genotyping

Single nucleotide polymorphisms (SNPs) are closely associated with many biological processes, including genetic disease, tumorigenesis, and drug metabolism. Accurate and efficient SNP determination has been proved pivotal in pharmacogenomics and diagnostics. Herein, a universal and high-fidelity genotyping platform is established based on the dual toeholds regulated Cas12a sensing methodology. Different from the conventional single stranded or double stranded activation mode, the dual toeholds regulated mode overcomes protospacer adjacent motif (PAM) limitation via cascade toehold mediated strand displacement reaction, which is highly universal and ultra-specific. To enhance the sensitivity for biological samples analysis, a modified isothermal recombinant polymerase amplification (RPA) strategy is developed via utilizing deoxythymidine substituted primer and uracil-DNA glycosylase (UDG) treatment, designated as RPA-UDG. The dsDNA products containing single stranded toehold domain generated in the RPA-UDG allow further incorporation with dual toeholds regulated Cas12a platform for high-fidelity human sample genotyping. We discriminate all the single-nucleotide polymorphisms of ApoE gene at rs429358 and rs7412 loci with human buccal swab samples with 100% accuracy. Furthermore, we engineer visual readout of genotyping results by exploiting commercial lateral flow strips, which opens new possibilities for field deployable implementation.

Human IgE-FcεRI chip assay to screen natural products for antipruritic activity

A biochip assay provides high-throughput and multiplexed analysis of biological samples, chemicals, and natural products. Pruritus is itchiness due to several causes, such as allergy, dry skin, pregnancy, liver disease, kidney disease, and thyroid disease. Treatment of pruritus is associated with reduced immunoglobulin (IgE) and FcεRI levels in sera. Treatment of pruritus include corticosteroid creams or ointment. However, they have side effects, so we need a safer treatment using natural products. Here, we developed an assay using protein chip technology to identify natural products with antipruritic activity that inhibit IgE-FcεRI binding. Of the 28 tested natural product extracts, Corni Fructus extract inhibited human IgE-FcεRI binding and also showed anti-histamine effects in MC/9 mast cells. These results suggest that this protein biochip assay system can be used to identify promising natural product extracts for the treatment of pruritus.

Analysis of vitamin D and its metabolites in biological samples – Part I: Optimization and comparison of UHPSFC-MS/MS and UHPLC-MS/MS methods

Fat-soluble vitamin D is an essential bioactive compound important for human health. Insufficient vitamin D levels can result not only in bone disease but also in other disorders, such as cancer, metabolic disorders, and diseases related to poor immune function. The current methods commonly used for vitamin D analysis are often applied to determine the levels of the most abundant metabolite in plasma, i.e., 25-OH-D2/D3. These methods do not consider the presence of other hydroxylated and esterified metabolites, including isomers and epimers, which are typically found in low concentrations.In this study, we developed a fast and selective ultra-high performance supercritical fluid chromatography (UHPSFC) method using a 150 mm long 1-amino anthracene (1-AA) column and a mobile phase consisting of carbon dioxide and methanol/isopropanol (1/1, v/v) mixed with 8 % water. After thorough optimization of column temperature and back pressure, the separation of four vitamin D3 esters, vitamin D3 and D2, and eight mono- and di-hydroxylated metabolites, including three groups of isomers, was achieved in 10 min. Two ion sources, atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization optimized within this study, were compared in tandem mass spectrometry (MS/MS) detection. No significant sensitivity differences were observed. Subsequently, the same 1-AA column chemistry was examined in ultra-high performance liquid chromatography (UHPLC) as the stationary phase that could hypothetically bring different selectivity in the separation of vitamin D and its metabolites. However, this hypothesis was rejected, and C18 was used as a stationary phase in the final optimized UHPLC-MS/MS method. Despite detailed optimization, the final 15 min UHPLC method was not able to separate di-hydroxylated isomers of vitamin D3, while it enabled better resolution of esterified forms compared to UHPSFC.Optimized methods provided similar repeatability of retention times and peak areas, with RSD < 2 % and 10 %, respectively. The lowest limits of quantification were in the range of 1.2 – 4.9 ng/mL for UHPSFC-APCI-MS/MS, while for UHPLC-APCI-MS/MS, they were typically in the range of 2.6 – 9.6 ng/mL. Based on the obtained results, the UHPSFC-APCI-MS/MS method was the most promising approach for fast, selective, and sensitive analysis that could be applied in the analysis of biological samples with emphasis on the separation of both hydroxylated and esterified metabolites, including isomeric forms.

Spontaneous Raman bioimaging – Looking to 2050

Raman imaging has been employed for a wide range of biological sample analyses, is often chosen for its non-invasiveness, and ability to provide rich information from samples with minimal preparation requirements. In this paper we give a brief overview of the applications of spontaneous Raman imaging in bioanalysis and then consider what Raman imaging in 2050 might look like. We discuss the state of Raman imaging around its inception, then provide a snapshot of current technology, then look towards 2050. We then discuss some of the potential bottlenecks for the continuing development of Raman imaging for biological sample analysis and, where appropriate, outline approaches to overcome these challenges.

Synthesis of biowaste-derived nanostructured material for the nanomolar detection of glucose: Biological sample analysis

Glucose electro-oxidation in an alkaline media was investigated using an electrode composed of carbon paste and sugarcane bagasse activated carbon (SBAC). The SBAC was prepared using an easy, affordable, and environmental friendly process. The porous activated carbon was produced using sugarcane bagasse through a pre-treatment process that involved carbonization at 650 °C and K2CO3. X-ray diffraction (XRD) was used to observe the formation of carbon crystallites during activation at higher temperatures. The Fourier-transform infrared (FTIR) spectrum shows functional groups that are present in the carbon material. The carbon sample's morphology was evaluated using scanning electron microscope (SEM). The surface roughness was studied using atomic force microscopy (AFM). Porous activated carbon derived from sugarcane bagasse was carefully mixed into electrode components such as graphite powder and mineral oil for glucose (GLS) detection. For precise GLS detection, the SBACs were employed as non-enzymatic electrochemical sensor probes in their as-prepared condition. According to the electrochemical experiments, the constructed sensor shows exceptional electrochemical performance towards glucose oxidation, including excellent selectivity, a low detection limit, good sensitivity, and a wide linear range. Investigation has been conducted on the oxidation of GLS in alkaline solutions with varying concentrations of halide ions such as iodide and chloride. The GLS oxidation peak current increased the performance of a SBAC modified electrode was compared to that of a bare carbon paste electrode (CPE). The role of sensitivity in minimizing halide poisoning is significant. The risk of poisoning is increased if iodide > chloride. As the modified electrode's voltage changes in a chloride solution containing glucose, the peak current gradually reduces. The redesigned electrode that is currently in use as a consequence not only promotes glucose oxidation but also shows strong resistance to electrode poisoning caused by halides. The sensitive and targeted SBAC/CPE has also improved its reliability while evaluating samples of human serum, urine, and breast milk.

Evaluation of stability of (1R,2 S)-(−)-2-methylamino-1-phenyl-1-propanol hydrochloride in plasma and urine samples-inoculated with Escherichia coli using high-performance liquid chromatography (HPLC)

The preservation of drug stability in biological evidence during the processes of collection and storage poses a substantial obstacle to the progress of forensic investigations. In conjunction with other constituents, the microorganisms present in the samples play a vital role in this investigation. The present investigation employed the high-performance liquid chromatography (HPLC) technique to assess the stability of (1R,2 S)-(–)-2-methylamino-1-phenyl-1-propanol hydrochloride in plasma and urine samples that were inoculated with Escherichia coli. These samples were subjected to storage conditions of 37 °C for 48 h and − 20 °C for a duration of 6 months. Minimal inhibitory concentration (MIC) and Minimal bactericidal concentration (MBC) of MPPH against E. coli were determined using microdilution method. The stability of MPPH in plasma and urine samples inoculated with E. coli was investigated using HPLC method. The results showed the MIC and MBC of MPPH were 87.5 ± 25 ppm and 175 ± 50 ppm, respectively. While MPPH remained stable in plasma for 48 h at 37 °C, it showed a notable decrease of about 11% in stability when stored in urine for the same period and temperature. From the beginning of the first month, a decrease in the stability of the compound appeared in all samples that were stored at − 20 °C, and the decrease reached 7% for plasma samples and about 11% for urine samples. The decrease in the stability reached its peak in the sixth month, reaching more than 30% and 70% of plasma and urine samples preserved at − 20 °C. This work concluded that E. coli can negatively affect the stability of MPPH in plasma and urine samples. This may lead to incorrect conclusions regarding the analysis of biological samples in criminal cases.

Sample preparation for fatty acid analysis in biological samples with mass spectrometry-based strategies.

Fatty acids (FAs) have attracted many interests for their pivotal roles in many biological processes. Imbalance of FAs is related to a variety of diseases, which makes the measurement of them important in biological samples. Over the past two decades, mass spectrometry (MS) has become an indispensable technique for the analysis of FAs owing to its high sensitivity and precision. Due to complex matrix effect of biological samples and inherent poor ionization efficiency of FAs in MS, sample preparation including extraction and chemical derivatization prior to analysis are often employed. Here, we describe an updated overview of FA extraction techniques, as well as representative derivatization methods utilized in different MS platforms including gas chromatography-MS, liquid chromatography-MS, and mass spectrometry imaging based on different chain lengths of FAs. Derivatization strategies for the identification of double bond location in unsaturated FAs are also summarized and highlighted. The advantages, disadvantages, and prospects of these methods are compared and discussed. This review provides the development and valuable information for sample pretreatment approaches and qualitative and quantitative analysis of interested FAs using different MS-based platforms in complex biological matrices. Finally, the challenges of FA analysis are summarized and the future perspectives are prospected.

Evaluation of an Oral Fluid Collection Device and a Solid-Phase Extraction Method for the Determination of Coca Leaf Alkaloids by Gas Chromatography-Mass Spectrometry.

Some South American countries have ancient traditions that may pose legal problems, such as the consumption of coca leaves, as this can provide positive results for cocaine use after the analysis of biological samples. For this reason, it is necessary to find specific markers that help differentiate legal from illegal consumption, such as tropacocaine, cinnamoylcocaine, and especially hygrine and cuscohygrine. In this work, two techniques for collecting biological samples are compared: the Quantisal® Oral Fluid collection device and passive drooling. Once the samples were collected, they were subjected to solid-phase extraction for subsequent injection into GC-MS. Different validation parameters included in international guides have been studied to evaluate whether the proposed method is valid for the defined purpose, placing special emphasis on the study of the matrix effect and little value on GC-MS analyses. With respect to this parameter, an increase in the signal was found for CUS and t-CIN, but it was not significant for the rest of the substances studied. The recoveries have varied significantly depending on the way of working, being higher when working with standardized areas. After carrying out work with the oral fluid samples collected from laboratory volunteers, the method was applied to two real samples. The results obtained support the need for further research to overcome certain limitations presented by the device.

An electrochemical biosensor based on DNA tetrahedron nanoprobe for sensitive and selective detection of doxorubicin

Doxorubicin (DOX) is a clinical chemotherapeutic drug and patients usually suffer from dose-dependent cytotoxic and side effects during chemotherapy process with DOX. Therefore, developing a reliable strategy for DOX analysis in biological samples for dosage guidance during chemotherapy process is of great significance. Herein, a sensitive and selective electrochemical biosensor for DOX detection was designed based on gold nanoparticles (AuNPs) and DNA tetrahedron (TDN) nanoprobe bifunctional glassy carbon electrode that could detect DOX in human serum and cell lysate samples. AuNPs not only could enhance electron transfer efficiency and detection sensitivity, but also could improve the biocompatibility of electrode. TDN nanoprobes were employed as specific DOX bind sites that could bind abundant DOX through intercalative characteristics to contribute to sensitive and selective detection. Under the optimal conditions, the proposed TDN nanoprobes-based DOX biosensor exhibited a wide linear range that ranged from 1.0 nM to 50 μM and a low detection limit that was 0.3 nM. Moreover, the proposed DOX biosensor displayed nice selectivity, reproducibility and stability, and was successfully applied for DOX detection in human serum and cell lysate samples. These promising results maybe pave a way for DOX dosage guidance and therapeutic efficacy optimization in clinic.

Direct Analysis of Whole Blood by a Disposable Monolithic Column Mass Spectrometry Analysis Kit†

Comprehensive SummaryA monolithic column‐based mass spectrometry (MS) analysis kit was prepared for whole blood analysis with MS. The kit is disposable and can be used for purification, storage, transportation and direct analysis of whole blood. The kit mainly consists of a capillary for quantitative microsampling, a cation exchange monolithic column for purification and storage, and a syringe for loading sample. This kit is very friendly to various users that one can easily siphon the blood in the kit followed by rapid clean‐up. We established a quantitative method using the kit with a limit detection as low as 0.33 nmol/L, and achieved more than five orders of magnitude enhancement in sensitivity compared to direct nanoelectrospray ionization MS analysis. The column can avoid analyte exposure to environment, which helps the storage of the sample for laboratory analysis. The relative standard deviation of immediate blood analysis and storage blood analysis within 10 d was less than 10%. This method has been successfully applied to the quantitative analysis of procainamide hydrochloride in 2 μL rat blood. These results indicate that this disposable kit does have the potential to achieve highly sensitive quantitative MS analysis in biological samples, which is expected to become a cost‐effective and powerful tool for in vitro diagnostics.