Evaluation of a one-step sample preparation protocol for analysis of total serum testosterone by LC–MS/MS
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PROVIDER: S-EPMC11322748 | biostudies-literature | 2018 Aug
REPOSITORIES: biostudies-literature
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Project description:Steroid analysis in clinical laboratories is dominated by immunoassays (IAs) that have a high sample turnover but are inherently limited in trueness, precision, and sensitivity. Liquid chromatography coupled to mass spectrometry (LC-MS/MS) has proved to be a far more capable tool, delivering better sensitivity, specificity, and the possibility of parallel analysis of multiple steroids and metabolites, providing the endocrinologist with more reliable and comprehensive diagnostic information. An LC-MS/MS assay with gradient elution over less than eight minutes and a one-step sample preparation combining protein precipitation with phospholipid removal of off-line solid-phase extraction was developed and validated. It allowed the quantification of 11-deoxycorticosterone (11-DOC), 11-deoxycortisol (11-DF), 17-OH-progesterone (17P), 21-deoxycortisol (21-DF), androstenedione (ANDRO), aldosterone (ALDO), corticosterone (CC), cortisol (CL), cortisone (CN), dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), dihydrotestosterone (DHT), estradiol (E2), progesterone (PROG), and testosterone (TES) in human serum. Interday imprecision was generally better than 15%, trueness was proven by recovery experiments with ISO 17034-certified reference materials, proficiency testing (UK NEQAS), and measuring serum reference standards. In-house comparison against IVD-CE-certified immunoassays (IA) for 17P, ANDRO, CL, DHEAS, E2, PROG, and TES was conducted by assessing leftover routine patient samples and purpose-built patient serum pools. None of the compared routine IAs were meeting the standards of the LC-MS/MS. Insufficient overall comparability was found for ANDRO and 17P (mean bias > +65%). Accuracy limitations at lower concentrations were present in IAs for PROG, E2, and TES.
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Project description:This protocol describes the peptidomic analysis of organoid lysates, FACS-purified cell populations, and 2D culture secretions by liquid chromatography mass spectrometry (LC-MS). Currently, most peptides are quantified by ELISA, limiting the peptides that can be studied. However, an LC-MS-based approach allows more peptides to be monitored. Our group has previously used LC-MS for tissue peptidomics and secretion of enteroendocrine peptides from primary culture. Now, we extend the use to organoid models. For complete details on the use and execution of this protocol, please refer to Goldspink et al. (2020).
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Project description:The sample condition is an important factor in urine proteomics with stability and accuracy. However, a general protocol of urine protein preparation in mass spectrometry analysis has not yet been established. Here, we proposed a workflow for optimized sample preparation based on methanol/chloroform (M/C) precipitation and in-solution trypsin digestion in LC-MS/MS-based urine proteomics. The urine proteins prepared by M/C precipitation showed around 80% of the protein recovery rate. The samples showed the largest number of identified proteins, which were over 1000 on average compared with other precipitation methods in LC-MS/MS-based urine proteomics. For further improvement of the workflow, the essences were arranged in protein dissolving and trypsin digestion step for the extraction of urine proteins. Addition of Ethylene diamine tetraacetic acid (EDTA) dramatically enhanced the dissolution of protein and promoted the trypsin activity in the digestion step because the treatment increased the number of identified proteins with less missed cleavage sites. Eventually, an optimized workflow was established by a well-organized strategy for daily use in the LC-MS/MS-based urine proteomics. The workflow will be of great help for several aims based on urine proteomics approaches, such as diagnosis and biomarker discovery.
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Project description:A very sensitive LC-MS/MS assay was developed implementing a liquid-liquid extraction step followed by mass spectrometry which was operated in both positive and negative ion modes. The assay was calibrated with readily available commercial calibrators and compared with international reference standards. This data is also presented in "Sensitive Simultaneous Quantitation of Testosterone and Estradiol in Serum by LC-MS/MS without Derivatization and Comparison with the CDC HoSt Program" (Schofield et al., 2017). This article includes the comparison of the LC-MS/MS assay with a commonly available chemiluminescencent immunoassay for the quantitation of both estradiol and testosterone. In addition we show baseline separation of estradiol and testosterone from other structurally related and/or isobaric compounds that could potentially interfere with the assay. In addition, various calibrator materials were tested and compared with internationally-recognized reference materials.
| S-EPMC6141700 | biostudies-literature
Project description:Background: Testosterone is an androgenic hormone that plays important roles in both males and females. The circulating levels of total testosterone vary from 1 to 1480 ng/dL. High-throughput immunoassays often lack accuracy in lower concentration ranges (below 100 ng/dL), particularly when used for females or children. To address this limitation, we developed a total testosterone LC-MS/MS assay on three instruments. Methods: Sample preparation began with the dilution and conditioning of 200 µL of serum. A supported liquid extraction cartridge was used to extract the analyte from biological matrices. Chromatographic separation was achieved using a C18 column with a runtime of 5 min per sample. This assay was validated on a Triple Quad 6500 and an API 4500 instrument. Results: Method validation was carried out according to the CLSI C62-ED2 guideline and our hospital protocol. The within-day coefficient of variation (CV) was less than 10% and the between-day CV was less than 15%. The assay had a limit of quantitation of 0.5 ng/dL with an analyte measure range of 2-1200 ng/dL. A comparison using Deming regression and Bland-Altman plots showed that this assay correlated well with a reference method. The results from the API 4500 and an Orbitrap were consistent with those from the TQ 6500. Both serum-separator tubes (BD) and serum-activator tubes were found to be suitable. Conclusions: We successfully developed and validated a robust total testosterone LC-MS/MS assay for routine clinical testing. This assay was harmonized across two triple quadrupole instruments and one high-resolution mass spectrometer.
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Project description:BackgroundHighly accurate and sensitive method to measure testosterone in hypogonadal male, female and children is vital for proper diagnosis of hormone-related conditions and their treatment.ObjectiveTo develop an accurate and robust total testosterone ESI-LC-MS/MS quantification method with a simple sample preparation workflow and sufficient sensitivity for serum or plasma samples of all gender and age groups, via ketone functional group derivatization (using Amplifex™ Keto Reagent).MethodA simple sample preparation method to accommodate both low and high numbers of samples was developed using simultaneous protein precipitation and derivatization with Amplifex™ Keto reagent, followed by centrifugation and direct injection of supernatant into an LC-MS/MS system (SCIEX Topaz™ IVD LC-MS/MS, in which MS is equivalent to a SCIEX 4500MD Mass Spectrometer). Total testosterone in human serum or plasma samples was quantified using an external calibration curve generated by calibrators spanning a broad concentration range of ∼1-2000 ng/dL (10-20,000 pg/mL), traceable to NIST 971 SRM. 13C3-enriched testosterone was used as an internal standard to correct for both analyte loss during sample preparation and matrix effect during analysis (Supplementary Information: SI Fig. 4C). Two methods, one using a 96-well filter plate and another using Eppendorf tubes, were developed. Both methods were certified by the Centers for Disease Control (CDC) hormone standardization (HoSt) program for total serum testosterone. The feasibility of implementing the method for plasma and serum samples was tested via a small-scale method comparison study between matched pediatric serum and plasma samples derived from the same donor. In addition, plasma samples originating from the same donor collected in two different anticoagulant tube types (Li-heparin and K2EDTA) were compared.ResultsUsing in-house formulated NIST 971-traceable calibrators, the method was linear (r2 > 0.999) between 1 and 2000 ng/dL (10 and 20,000 pg/mL) with a limit of detection of approximately 1 ng/dL (10 pg/mL). The testosterone concentration bias against 40 reference samples from the HoSt certification program was absolute <3% with an average %CV of ∼3-4%. More than 78% of samples passed the CDC bias criterion of ±6.4%. Comparison between pediatric matched serum and plasma samples resulted in high correlation (r2 = 0.997) and bias of <5%. The calculated % difference between matched adult serum and plasma samples was ∼1%.ConclusionsFeasibility for an accurate and streamlined method suitable for measuring total testosterone in all human samples was demonstrated with a choice of sample preparation workflow to suit low or high number of samples. The method can potentially be used for plasma matrix from different blood collection tubes (Li-Heparin and K2EDTA).
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Project description:The detection and quantification of nut allergens remains a major challenge. The liquid chroma-tography tandem mass spectrometry (LC-MS/MS) is emerging as one of the most widely used methods, but sample preparation prior to the analysis is still a key issue. The objective of this work was to establish optimized protocols for extraction, tryptic digestion and LC-MS analysis of almond, cashew, hazelnut, peanut, pistachio and walnut samples. Ammonium bicar-bonate/urea extraction (Ambi/urea), SDS buffer extraction (SDS), polyvinylpolypyrroli-done (PVPP) extraction, trichloroacetic acid/acetone extraction (TCA/acetone) and chloro-form/methanol/sodium chloride precipitation (CM/NaCl) as well as the performances of con-ventional tryptic digestion and microwave-assisted breakdown were investigated. Overall, the protein extraction yields ranged from 14.9 ± 0.5 (almond extract from CM/NaCl) to 76.5 ± 1.3% (hazelnut extract from Ambi/urea). Electrophoretic profiling showed that the SDS extraction method clearly presented a high amount of extracted proteins in the range of 0-15 kDa, 15-35 kDa, 35-70 kDa and 70-250 kDa compared to the other methods. The linearity of the LC-MS methods in the range of 0 to 0.4 µg equivalent defatted nut flour was assessed and recovery of internal standards GWGG and DPLNV(d8)LKPR ranged from 80 to 120%. The identified bi-omarkers peptides were used to relatively quantifier selected allergenic protein form the inves-tigated nut samples. Considering the overall results, it can be concluded that SDS buffer allows a better protein extraction from almond, peanut and walnut samples while PVPP buffer is more appropriate for cashew, pistachio and hazelnut samples. It was also found that conventional overnight digestion is indicated for cashew, pistachio and hazelnut samples, while microwave assisted tryptic digestion is recommended for almond, hazelnut and peanut extracts.
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