BACKGROUND: To date, clinical laboratories face challenges in quantifying retinol from DBS samples. Disputes arise throughout the whole detection process, encompassing the storage condition, the release strategy as well as the selection of internal standards.
METHODS: We incubated DBS with ascorbic acid solution. Then, retinol-d4 in acetonitrile was introduced to incorporate isotopic internal standard and promote protein precipitation. Afterward, sodium carbonate solution was added to ionize cytochromes (such as bilirubin), which amplified the difference of their hydrophobicity to retinol. Subsequently, cold-induced phase separation could be facilitated to separate retinol from the impurities. In the end, the upper layer was injected for LC-MS/MS analysis.
RESULTS: By comparing the detected retinol content in whole blood and DBS samples prepared from the same volume, we confirmed the established pretreatment was capable to extract most of retinol from DBS (recovery >90 %). Thereafter, we verified that within DBS, retinol possessed satisfying stability without antioxidation. Indoor-light exposure and storage duration would not cause obvious degradation (<10 %). Following systematic validation, the established method well met the criteria outlined in the relevant guidelines. After comparing with detected DBS results to the paired plasma samples, 54 out of 60 met the acceptance limit for cross-validation of ±20 %.
CONCLUSIONS: We realized precise quantification of retinol from one 3.2 mm DBS disc. By circumventing conventional antioxidation, liquid-liquid/solid-phase extraction and organic solvent evaporation, the pretreatment could be completed within 15 min consuming only minimal amounts of low-toxicity chemicals (ascorbic acid, acetonitrile, and sodium carbonate). We expect this contribution holds the potential to significantly facilitate the evaluation of patients\' vitamin A status by using DBS samples in the future.

Alternaria toxins are naturally occurring contaminants found in natural products. Given the prevalence of Alternaria toxins and the complexity of oil-rich matrices, achieving ultra-trace analysis has become a daunting task. A new sample pretreatment technique, i.e., cold-induced liquid-liquid microextraction combined with serially-coupled-columns for SIDA-UHPLC-MS/MS, was developed and reported for the first time. Theoretical and experimental investigations on the mechanism and key parameters revealed that the proposed method achieved simultaneous purification and enrichment in one-step sample extraction with a superior limit of quantitation (0.15-1.5 μg kg-1), without further sample manipulation, such as fat removal or solvent exchange procedures prior to LC-MS. The method was validated taking into consideration EU guidelines and showed acceptable linearity (r ≥ 0.9991), accuracy with recoveries between 75 and 114% and precision with RSD≤9.7% for all of the analytes studied. It was successfully applied to the analysis of twenty samples sourced from the Mediterranean region in order to gain first insights into Alternaria toxins contaminations in olive oils. This technical approach is well suited for large-scale studies in a high-throughput and cost-effective quality assurance laboratory environments, and it has the potential to detect ultra-trace levels of toxins in complex samples, which may lead to the development of new and sustainable sample preparation procedures.

Dried blood spots (DBS) have been regarded as a promising alternative for therapeutic drug monitoring (TDM) of immunosuppressants (ISDs) for over fifteen years. Nonetheless, there are still three main issues impeding its preference: (i) the requirement of relatively large disc; (ii) the controversial and intricate desorption approaches; (iii) the lack of feasible extraction strategies. For improvement, this work described a new LC-MS/MS method realizing quantification of four ISDs from one piece of 3.2 mm DBS. During sample pretreatment, a modified approach (infiltrating the DBS in pure water before adding acetonitrile and zinc sulfate as protein-precipitators) was developed to completely dissociate the targets from filter paper. Afterward, effective enrichment and purification of the targets were achieved by using cold-induced phase separation technique. Benefiting from these novelties, the method exhibited satisfying throughput (15 min for sample preparation), applicability (consuming only one 3.2 mm disc), reliability (82.3-107.8% for accuracy and <14.3% for precision) and sensitivity (lower limit of quantification of 0.5, 7.6, 0.7 and 0.8 ng mL-1 for tacrolimus, cyclosporine A, everolimus and sirolimus, respectively). Without hematocrit correction, the method showed favorable interchangeability to the certified whole blood method through analyzing 120 paired clinical samples. By taking ±20% of the mean as the limit of acceptance for cross validation, over 90% of the detection met the criterion. It can be expected the developed method is able to further promote the popularization of DBS-based TDM for ISDs in practice.

Sex hormones, including androgens, estrogens, and progestogens, are important biomarkers for various diseases. Quantification of sex hormones is typically conducted by LC-MS/MS. At present, most methods require liquid-liquid extraction or solid phase extraction for sample preparation. However, these pretreatments are prone to compromise LC-MS/MS throughput. To improve on the current standard practices, we investigated cold-induced phase separation for sex hormone extraction. After protein precipitation with acetonitrile and adjusting the solution constitution with water, samples were stored at -30°C for 10 min to generate two distinct phases: an acetonitrile-rich layer on top of a water-rich layer. During this process, the hydrophobic sex hormones spontaneously separate into the upper layer. This simple and reliable cold-induced phase separation-based LC-MS/MS methodology was used here to simultaneously detect estrone, estradiol, estriol, testosterone, androstenedione, dehydroepiandrosterone, progesterone, and 17-hydroxyprogesterone in serum. Validation of this method indicated satisfactory performance, including acceptable linearity, accuracy, precision, and tractability. Compared with the mainstream liquid-liquid extraction-based method, this new method exhibits significant progress in throughput, which shortens the time cost of sample preparation from 90 to 40 min. We propose that this method can be an excellent alternative for sex hormone analysis in routine clinical laboratories.

Extensive residues of neonicotinoids (neonics) have been demonstrated in food and the environment by routine monitoring measurement, but little is known about the residue levels in \"ready to eat\" dietary samples. To obtain a more accurate picture of dietary exposure to total neonics, an ultrasensitive and effective cleanup analytical method for the quantification of neonics in dietary samples was established on the basis of cold-induced phase separation and pre-column dilution injection liquid chromatography-high-resolution mass spectrometry. A total of 10 neonics were quantified in ultratrace amounts (ng/kg) using stable isotope dilution, with calibration curves spanning 4 orders of magnitude. Satisfactory accuracy (73.5-109.2% for the recoveries) and precision (0.6-13.2% for the relative standard deviation ranges) were obtained in method validation. Moreover, tolerable absolute matrix effects (0.89-1.09) were also obtained in 12 kinds of dietary matrices with weak relative matrix effects (2.8-12.6%). The validated method was applied to the dietary samples collected from the Chinese Total Diet Study, and the results showed that 75% of the samples were contaminated with at least one neonicotinoid.

Recently, the phenomenon of acute poisoning events caused by glyphosate (GLY) had frequently occurred all over the world. The present work reported a simple liquid chromatography-high resolution mass spectrometry (LC-HRMS) method for direct determination of GLY and its metabolite aminomethylphosphonic acid (AMPA) in human urine by combining cold-induced phase separation (CIPS) with hydrophilic pipette tip solid-phase extraction (PTSPE). First, a urine sample was mixed with acetonitrile at a 80% concentration to precipitate proteins. After centrifugation, the mixture was performed a CIPS at -20 °C to enrich GLY and AMPA (six-fold) in the lower water phase which was further performed PTSPE procedure. PTSPE as a miniaturized procedure of SPE, combined with a manual accu-jet® Pro Pipette Controller, was used to extract GLY and AMPA, in which a new type of hydrophilic adsorbent (HILIC powder) based on amide-modified silica was selected as the adsorption of GLY and AMPA. The key factors including the type and the amount of adsorbent, the loading extraction solution, the type and volume of eluent, and the number of aspirating/dispensing cycles were investigated in detail. Meanwhile, the selectivity and sensitivity of GLY and AMPA analysis were improved by the use of LC-HRMS based on targeted single ion monitoring (tSIM) mode without tedious derivatization. This method made a full use of the advantages of these techniques by combining efficient enrichment, effective extraction and selective separation in a simple way. Finally, a comprehensive validation of the method was rigorously executed and the results indicated that the validated method afforded desired linearity, precision, accuracy, and sensitivity.