Beibei Zhao

With time, the number of samples in clinical laboratories from therapeutic drug monitoring has increased. Existing analytical methods for blood cyclosporin A (CSA) monitoring, such as high‑performance liquid chromatography (HPLC) and immunoassays, have limitations including cross‑reactivity, time consumption, and the complicated procedures involved. Liquid chromatography‑tandem mass spectrometry (LC‑MS/MS) has long been considered the reference standard owing to its high accuracy, specificity, and sensitivity. However, large numbers of blood samples, multi‑step preparation procedures, and longer analytical times (2.5‑20 min) are required as a consequence of the different technical strategies, to ensure good analytical performance and routine quality assurance. A stable, reliable, and high throughput detection method will save personnel time and reduce laboratory costs. Therefore, a high throughput and simple LC‑MS/MS method was developed and validated for the detection of whole‑blood CSA with CSA‑d12 as the internal standard in the present study. Whole blood samples were prepared through a modified one‑step protein precipitation method. A C18 column (50x2.1 mm, 2.7 µm) with a mobile phase flow rate of 0.5 ml/min was used for chromatographic separation with a total running time of 4.3 min to avoid the matrix effect. To protect the mass spectrometer, only part of the sample after LC separation was allowed to enter the mass spectrum, using two HPLC systems coupled to one mass spectrometry. In this way, throughput was improved with detection of two samples possible within 4.3 min using a shorter analytical time for each sample of 2.15 min. This modified LC‑MS/MS method showed excellent analytical performance and demonstrated less matrix effect and a wide linear range. The design of multi‑LC systems coupled with one mass spectrometry may play a notable role in the improvement of daily detection throughput, speeding up LC‑MS/MS, and allowing it to be an integral part of continuous diagnostics in the near future.

Urinary and plasma 18-hydroxycortisol (18-OHF) have been investigated for primary aldosteronism (PA) subtyping. However, there is no research exploring the impact of sample types on the diagnostic performance of 18-OHF in PA subtyping. In this study, 18-OHF levels in both urine and plasma were determined in patients with idiopathic adrenal hyperplasia (IHA), aldosterone-producing adenoma (APA), and essential hypertension (EH) by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Urinary18-OHF was determined using an established LC-MS/MS method, whereas plasma18-OHF was measured by a modified LC-MS/MS method. Differences in urinary and plasma 18-OHF levels between APA, IHA, and EH patients were investigated by Kruskal-Wallis test for non-parametric analysis. The LC-MS/MS method yielded a lower limit of quantitation (LLOQ) of 18-OHF in urine of 4.28 nmol/L and 0.190 nmol/L in plasma. The intra- and inter-precision for urine and plasma methods were < 6%, with accuracies between 95.9% and 110.3%. Urinary and plasma 18-OHF in 12 IHA, 18 APA, and 91 EH patients were quantified and analyzed. Non-parametric analysis by Kruskal-Wallis test revealed that urinary 18-OHF levels in IHA and APA patients were significantly different (P < 0.05) while plasma 18-OHF levels were not (P > 0.05), indicating that urinary 18-hydroxycortisol outperformed plasma 18-hydroxycortisol for primary aldosteronism subtyping.

Purpose We aimed to describe the vitamin D status and its distribution in different age groups, sexes, seasons, and provinces of a large Chinese population. Methods This study retrospectively analyzed 1,528,685 results of serum 25-hydroxyvitamin D (25(OH)D) in the central laboratory of KingMed Diagnostics. The samples were from the individuals aged 0–119 years old in 30 provinces of China. Serum 25(OH)D was measured by an accurate commercial liquid chromatography-tandem mass spectrometry (LC–MS/MS) method from January 2017 to December 2019. The subjects were stratified by age, sex, the season of blood collection, and the province of residence. Results The median 25(OH)D concentration was 25.5 ng/mL (interquartile range (IQR) 18.7–32.7 ng/mL) in males and 20.8 ng/mL (IQR 14.4–28.2 ng/mL) in females. Overall, the median 25(OH)D concentration decreased with age in both males and females. Males had a 0.2–2.4 ng/mL higher median 25(OH)D concentration than females in different age groups. Vitamin D deficiency (25(OH)D < 15 ng/mL for the individuals under 14 years old; < 20 ng/mL for the individuals over 14 years old) was found in 21.3% of males and 43.6% of females. Significant seasonal variation of serum 25(OH)D concentrations was repeatedly observed in 3 years, with median concentration higher in summer (25.3 ng/mL (IQR 19.3–31.9 ng/mL)) and lower in winter (18.5 ng/mL (IQR 12.3–26.6 ng/mL)). Vitamin D status varied by province. The median 25(OH)D concentration was the highest in Hainan (31.0 ng/mL (IQR 24.9–39.2 ng/mL)) and the lowest in Qinghai (14.4 ng/mL (IQR 9.6–20.0 ng/mL)). 25(OH)D₂ was detected in 12.2% of the results, and no significant seasonal variation was observed. Conclusion In China, vitamin D deficiency is prevalent in the population participating in clinical vitamin D measurement. Age and sex differences in vitamin D levels were observed in our study. Seasonal variation and provincial differences are important aspects of serum vitamin D status. 25(OH)D₂ cannot be ignored entirely in clinical measurement practice in China.

The eicosanoid metabolic pathway is responsible for mediating the production of various inflammatory factors that are closely related to the development and resolution of inflammation. In biological matrices, the major quantifying obstacles were shown to be the oxidation and low quantities of eicosanoids and their metabolites. This study aimed to develop a reliable, sensitive ultrahigh-performance liquid chromatography coupled to a tandem mass spectrometry (UPLC–MS/MS) method to quantify eicosanoids in human serum. Solid-phase extraction (SPE) was used for sample preparation. The approach employed continuous ionization polarity switching. The target eicosanoids showed good linearity over the investigated concentration range (r2 > 0.99). The recovery rates were over 64.5%, and the matrix effects ranged from 73.0 to 128.0%. The limits of quantification were 0.048 ~ 0.44 ng/mL. For the broad concentration range, the CV % for accuracy and precision were less than ± 20%. We successfully applied this method to rapidly analyse 74 serum samples from severe influenza pneumonia, severe bacterial pneumonia and healthy individuals. Eicosanoid-related metabolite concentrations were quantified within a range similar to those of previously published articles. Compared to healthy individuals, our application found that 20-HETE, 14,15-EET and 11,12-EET were upregulated in severe influenza pneumonia patients, while LTB₄ was downregulated. 8-HETE and 5-HETE were upregulated in severe bacterial pneumonia patients, while LTE₄ was downregulated. This approach provides a means for monitoring the low quantities of eicosanoids in biological matrices, and our finding that different characteristic metabolite profiles may help discriminate the induction of severe pneumonia patients.

Background: Eighteen‐hydroxycortisol (18‐OHF) is a potential biomarker for differential diagnosis of the two major primary aldosteronism subtypes, aldosterone‐producing adenoma, and idiopathic hyperaldosteronism.Methods: Urine samples were processed, and the 18‐OHF in urine samples were successfully quantified by in‐house established dilute‐and‐shoot liquid chromatography–tandem mass spectrometry (LC–MS/MS) method. Separation was accomplished on a Sigma Ascentis Express C18 column with a gradient mixture of phase (A) 0.2% formic acid in water and phase (B) 0.2% formic acid in methanol at a flow rate of 0.4 ml/min. Mass spectrometric detection was performed in positive electrospray ionization mode via a mass spectrometer.Results: The linearity of urinary 18‐OHF ranged from 4.28 to 8.77 × 10³ nmol/L, with a lower limit of quantification at 4.28 nmol/L. The intra‐ and inter‐precision were both below 3%. The range of analytical recovery was 97.8%–109.2%. The validated dilute‐and‐shoot LC–MS/MS method was compared with the SPE LC–MS/MS method modified from the one reported in 2013. The results by Passing–Bablok regression analysis and Bland–Altman plotting demonstrated a good agreement between the two methods. The presented method was then applied to establish sex‐specific reference intervals from 62 males and 62 females, respectively. The calculated 2.5%–97.5% reference intervals for 24‐h urinary 18‐OHF were 113–703 nmol/day for males and 71.2–450 nmol/day for females.Conclusion: The presented dilute‐and‐shoot LC–MS/MS method for 18‐OHF quantification showed a good performance in the clinical application. Furthermore, the sex‐specific reference intervals for 24‐h urinary 18‐OHF were first established and quite important for its application in primary aldosteronism subtyping.

Backgrounds: Systemic amyloidosis is classified according to the deposited amyloid fibril protein (AFP), which determines its best therapeutic scheme. The most common type of AFP found are immunoglobulin light chains. The laser microdissection combined with mass spectrometry (LMD-MS) technique is a promising approach for precise typing of amyloidosis, however, the major difficulty in interpreting the MS data is how to accurately identify the precipitated AFP from background. Objectives: The objective of the present study is to establish a complete data interpretation procedure for LMD-MS based amyloidosis typing. Methods: Formalin-fixed paraffin-embedded specimens from patients with renal amyloidosis and non-amyloid nephropathies (including diabetic nephropathy, fibrillary glomerulonephritis, IgA nephropathy, lupus nephritis, membranous nephropathy, and normal tissue adjacent to tumors) were analyzed by LMD-MS. Forty-two specimens were used to train the data interpretation procedure, which was validated by another 50 validation specimens. Area under receiver operating curve (AUROC) analysis of amyloid accompanying proteins (AAPs, including apolipoprotein A-IV, apolipoprotein E and serum amyloid P-component) for discriminating amyloidosis from non-amyloid nephropathies was performed. Results: A stepwise data interpretation procedure that includes or excludes the types of amyloidosis group by group was established. The involvement of AFPs other than immunoglobulin was determined by P-score, as well as immunoglobulin light chain by variable of λ-κ, and immunoglobulin heavy chain by H-score. This achieved a total of 88% accuracy in 50 validation specimens. The AAPs showed significantly different expression levels between amyloidosis specimens and non-amyloid nephropathies. Each of the single AAP had a AUROC value more than 0.9 for diagnosis of amyloidosis from non-amyloid control, and the averaged level of the three AAPs showed the highest AUROC (0.966), which might be an alternative indicator for amyloidosis diagnosis. Conclusions: The proteomic data interpretation procedure for LMD-MS based amyloidosis typing was established successfully that has a high practicability in clinical application.

Backgrounds: Systemic amyloidosis is classified according to the deposited amyloid protein, which determines its best therapeutic scheme. The laser microdissection combined with mass spectrometry (LMD-MS) technique is a promising approach for precise subtyping of amyloidosis, however, is hampered by how to interpret the MS data.Objectives: The objective of the present study is to establish a complete data interpretation procedure for LMD-MS based amyloidosis subtyping.Methods: Formalin fixed paraffin-embedded specimens from patients with renal amyloidosis were analyzed by LMD-MS for proteome quantification. Forty-two specimens were used for training the data interpretation procedure, which was validated by another 50 validation specimens. Area under receiver operating curve (AUROC) analysis of amyloid accompanying proteins (APOE, APOA4 and SAMP) for discriminating amyloidosis from non-amyloid nephropathies was performed.Results: A stepwise data interpretation procedure that include or exclude the subtypes group by group was established, in which, involvement of non-immunoglobulin amyloid protein is determined by P-score, involvement of immunoglobulin light chain is determined by variable of λ-κ, and immunoglobulin heavy chain’s participation is judged by H-score. This data interpretation method achieved a 88% accuracy in 50 validation specimens. The amyloid accompanying proteins showed significant quantitative differences between amyloidosis specimens and non-amyloid nephropathies. Each of the single accompanying protein had a AUROC value more than 0.9 for diagnosis of amyloidosis from non-amyloid control, and the averaged value of spectral count of the three accompanying proteins showed the highest AUROC (0.966), indicating it might be an alternative indicator for amyloidosis diagnosis.Conclusions: The proteomic data interpretation procedure for amyloidosis subtyping based on LMD-MS was established successfully, which has high clinical application value.