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Proteins are essential components of human cells and tissues, and they are commonly measured in clinical laboratories using immunoassays. However, these assays have certain limitations, such as non-specificity binding, insufficient selectivity, and interference of antibodies. More sensitive, accurate, and efficient technology is required to overcome these limitations. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a powerful analytical tool that provides high sensitivity and specificity, making it superior to traditional methods such as biochemical methods and immunoassays. While LC-MS/MS has been increasingly used for detecting small molecular analytes and steroid hormones in clinical practice recently, its application for protein or peptide analysis is still in its early stages. Established methods for quantifying proteins and peptides by LC-MS/MS are mainly focused on scientific research, and only a few proteins and peptides can be or have the potential to be detected and applied in clinical practice. Therefore, this article aims to review the clinical applications, advantages, and challenges of analyzing proteins and peptides using LC-MS/MS in clinical laboratories., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Vitamin D deficiency is common in pregnancy, however, its effects has not been fully elucidated. Here, we conducted targeted metabolomics profiling to study the relationship., Methods: This study enrolled 111 pregnant women, including sufficient group (n = 9), inadequate group (n = 49) and deficient group (n = 53). Ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS)-based targeted metabonomics were used to characterize metabolite profiles associated with vitamin D deficiency in pregnancy., Results: Many metabolites decreased in the inadequate and deficient group, including lipids, amino acids and others. The lipid species included fatty acyls (FA 14:3, FA 26:0; O), glycerolipids (MG 18:2), glycerophospholipids (LPG 20:5, PE-Cer 40:1; O2, PG 29:0), sterol lipids (CE 20:5, ST 28:0; O4, ST 28:1; O4). Decreased amino acids included aromatic amino acids (tryptophan, phenylalanine, tyrosine) and branched-chain amino acids (valine, isoleucine, leucine), proline, methionine, arginine, lysine, alanine, L-kynurenine,5-hydroxy-L-tryptophan, allysine., Conclusions: This targeted metabolomics profiling indicated that vitamin D supplementation can significantly affect lipids and amino acids metabolism in pregnancy., (© 2024. The Author(s).)

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has attracted significant attention in clinical practice owing to its numerous advantages. However, the widespread adoption of this technique is hindered by certain limitations, such as inappropriate analyte selection, low levels of automation, and a lack of specific reference intervals and quality control programs. This review comprehensively summarizes the current challenges associated with LC-MS/MS and proposes potential resolutions. The principle of utility should guide the selection of biomarkers, prioritizing their practical value over sheer quantity. To achieve full-process automation, methodological innovation is crucial for developing high-throughput equipment. Establishing reference intervals for mass spectrometry-based assays across multiple centers and diverse populations is essential for accurate result interpretation. Additionally, the development of commercial quality control materials assumes pivotal importance in ensuring assay reliability and reproducibility. Harmonization and standardization efforts should focus on the development of reference methods and materials for the clinical use of LC-MS/MS. In the future, commercial assay kits and laboratory-developed tests (LDTs) are expected to coexist in clinical laboratories, each offering distinct advantages. The collaborative efforts of diverse professionals is vital for addressing the challenges associated with the clinical application of LC-MS/MS. The anticipated advancements include simplification, increased automation, intelligence, and the standardization of LC-MS/MS, ultimately facilitating its seamless integration into clinical routines for both technicians and clinicians., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Traditional solid-phase extraction (SPE) LC-MS/MS is limited by high costs, turnaround times, and procedural complexity, which limited the usage in clinical practice. This study aimed to establish a robust UPLC-MS/MS method with automated magnetic-bead-assisted sequential extraction (MBASE) technology to simultaneously measure Aβ1-42 and Aβ1-40 in cerebrospinal fluid (CSF). A Waters TQ-XS triple quadrupole mass spectrometer and Acquity UPLC Protein BEH C4 column were used. The targeted analytes were extracted and concentrated using the automated MBASE technology with chemically modified magnetic MCX beads. Analytical performance was verified referring to the CLSI C62-A and EP-15-A3 guidelines. A total of 68 CSF samples were collected and analyzed using the MBASE UPLC-MS/MS method, traditional SPE UPLC-MS/MS method, and Lumipulse G fully automated chemiluminescence detection system, and method comparison analysis is conducted. The MBASE UHPLC-MS/MS method showed an analytical performance equivalent to that of traditional SPE technology, with a higher sample throughput and smaller amount of materials ($34.98 vs. $493.96) and labor cost (101 min vs. 140 min) for 96 samples. The limit of quantification (LOQ) of Aβ1-42 and Aβ1-40 was 0.10 ng/mL and 0.05 ng/mL; recovery was 88.35-107.07 % and 95.72-96.60 %; and total imprecision was 3.69-6.83 % and 3.02-3.61 %, respectively. The measurements were faithfully reproduced within the allowable levels of uncertainty using certified reference materials. The correlations between this MBASE UPLC-MS/MS method, the SPE UPLC-MS/MS method, and Lumipulse G fully automated biochemical analysis method are all deemed good (r = 0.869-0.936), and the MBASE- and SPE-UPLC-MS/MS methods showed comparable measurements. To our knowledge, our study firstly verified the robust performance of the MBASE UPLC-MS/MS method to simultaneously determine Aβ1-42 and Aβ1-40 in CSF. With further introduce of automation, the assay with high accuracy and low material and labor costs will become a promising clinical technology., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have ppeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Steroids are essential in the differential diagnosis of congenital adrenal hyperplasia (CAH) subtypes; however, they may confuse physicians with multifarious results. In this study, we established a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous measurement of 24 steroids and developed a steroid metabolite pathway-based report to aid physicians in understanding these results. Solid-phase extraction was used to concentrate and purify target plasma steroids. The linearity, precision, recovery, and matrix effects were thoroughly evaluated. PowerBuilder was used to transfer the results from LC-MS/MS to the graphic report in a laboratory information management system (LIS) and was applied to different subtypes of CAH. Twenty-four steroids were separated and analyzed in one sample preparation and two injections using LC-MS/MS. The linearity of the steroids was excellent, with coefficients of linear regression greater than 0.99. The relative recovery ranged from 90.0 to 107.1 %, whereas the intra- and total coefficient variations were 1.6 ∼ 8.7 % and 2.0 ∼ 9.9 %, respectively. Matrix effects were compensated after internal standard correction. A graphic combination report mode was established and used to effectively identify CAH subtypes. In conclusion, a useful LC-MS/MS method and graphic combination report of 24 steroids based on their metabolite pathways were established., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Fat-soluble vitamins, including vitamins A, D, E, and K, are essential for maintaining normal body function and metabolism. Fat-soluble vitamin deficiency may lead to bone diseases, anemia, bleeding, xerophthalmia, etc. Early detection and timely interventions are significant for preventing vitamin deficiency-related diseases. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is developing into a potent instrument for the precise detection of fat-soluble vitamins due to its high sensitivity, high specificity, and high resolution., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: Regular sleep is very important for human health; however, the short-term and long-term effects of nightshift with sleep deprivation and disturbance on human metabolism, such as oxidative stress, have not been effectively evaluated based on a realistic cohort. We conducted the first long-term follow-up cohort study to evaluate the effect of nightshift work on DNA damage., Methods: We recruited 16 healthy volunteers (aged 33 ± 5 years) working night shifts at the Department of Laboratory Medicine at a local hospital. Their matched serum and urine samples were collected at four time points: before, during (twice), and after the nightshift period. The levels of 8-oxo-7,8-dihydroguanosine (8-oxoG) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), two important nucleic-acid damage markers, were accurately determined based on a robust self-established LC‒MS/MS method. The Mann-Whitney U or Kruskal-Wallis test was used for comparisons, and Pearson's or Spearman's correlation analysis was used to calculate the correlation coefficients., Results: The levels of serum 8-oxodG, estimated glomerular filtration rate-corrected serum 8-oxodG, and the serum-to-urine 8-oxodG ratio significantly increased during the nightshift period. These levels were significantly higher than pre-nightshift work level even after 1 month of discontinuation, but no such significant change was found for 8-oxoG. Moreover, 8-oxoG and 8-oxodG levels were significantly positively associated with many routine biomarkers, such as total bilirubin and urea levels, and significantly negatively associated with serum lipids, such as total cholesterol levels., Conclusion: The results of our cohort study suggested that working night shifts may increase oxidative DNA damage even after a month of discontinuing nightshift work. Further studies with large-scale cohorts, different nightshift modes, and longer follow-up times are needed to clarify the short- and long-term effects of night shifts on DNA damage and find effective solutions to combat the negative effects., (© 2023. The Author(s).)

Background: Examination of aldosterone to Renin Ratio (ARR) and plasma aldosterone concentration (PAC) or 24-h urinary aldosterone excretion (24-h UALD) was the necessary tests in confirmatory tests for primary aldosteronism (PA). We developed a combined liquid chromatography-tandem mass spectrometry method (LC-MS/MS) for plasma renin activity (PRA), PAC, and angiotensin II (Ang II) and investigated their reference intervals (RIs) in northern Chinese Han population. The RIs of 24-h UALD excretion were also studied using LC-MS/MS., Methods: A total of 309 healthy volunteers were recruited in 3 cities in China. PRA, PAC, Ang II, and 24-h UALD were measured using the laboratory-developed LC-MS/MS. Multiple linear regression and the variance component model were applied to determine if the RI needed to be split. The RIs of PRA, PAC, and Ang II were determined using the nonparametric percentile method., Results: The laboratory-developed LC-MS/MS method was verified and showed good performance. Standard deviation ratio (SDR) sex for PAC and SDR region for Ang II are 0.466 and 0.407, respectively, indicating that the RIs of PAC and Ang II must be divided by sex and region, respectively. In addition, the SDR 24hUK for 24-h UALD is 0.579, indicating that the RI of 24-h UALD must be partitioned by urine potassium., Conclusion: RIs were established for tests related to the renin-angiotensin-aldosterone system in the apparently healthy northern Chinese Han population by the LC-MS/MS method., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Sample preparation is the rate-limiting step in liquid chromatography-mass spectrometry (LC-MS)/MS-based clinical analysis when target analytes possess significantly different properties. Repeated solid-phase extraction (SPE) processes are typically required, resulting in low throughput and excessive consumption of labor, materials, and samples. In this study, we developed and validated a feasible and productive method to enrich target analytes with different properties during a single operation, while sufficiently removing matrix interferences to meet LC-MS/MS requirements. Gastrin determination was selected as the subject of this study. An automated magnetic-bead-assisted sequential extraction (MBASE) workflow was developed to simultaneously isolate nonsulfated gastrin-17 (G17ns), sulfated gastrin-17 (G17s), nonsulfated gastrin-34 (G34ns), and sulfated gastrin-34 (G34s) from human serum. It performs two different ion-exchange-based magnetic-bead extraction steps on one sample aliquot to produce one combined extract for LC-MS/MS analysis. When compared with the traditional SPE process, the MBASE workflow saves over 75% time and labor expenses as well as over 90% material cost, while providing even higher extraction efficiency. The MBASE LC-MS/MS method was validated as accurate and robust. Clinical sample test results demonstrated that the conventional chemiluminescence immunoassay method significantly under-estimated total gastrins in human serum, and the MBASE LC-MS/MS method could serve as an ideal tool to provide a comprehensive and accurate gastrin profile.

Objective: To establish and validate a robust LC-MS/MS method for simultaneously measuring 8-oxoGuo, 8-oxodG, and NMN in serum and urine to evaluate the oxidative stress status., Methods: A Waters TQ-XS triple quadrupole mass spectrometer system coupled with an Acquity UPLC Primer HSS T3 column was chosen. The clinical performance was verified according to the CLSI C62-A and EP-15 guidelines. Furthermore, matched serum and urine samples from 22 apparently healthy check-ups, 20 patients with atherosclerosis, and 18 individuals with dementia were evaluated., Results: The recovery for serum 8-oxoGuo, urine 8-oxoGuo, serum 8-oxodG, urine 8-oxodG, serum NMN, and urine NMN was 88.8-112.4%, 102.4-114.1%, 88.5-107.7%, 94.9-102.6%, 98.4-108.9%, and 88.5-108.6%, respectively. Based on the inter-assay results, total coefficient of variation, matrix effect, and carryover, the LC-MS/MS method was deemed robust. The limit of quantification was 0.017, 0.018, and 0.150 nmol/L for 8-oxoGuo, 8-oxodG, and NMN, respectively, which are suitable for accurate measurements in human serum and urine samples. Higher 8-oxoGuo and 8-oxodG levels and lower NMN levels, indicative of significantly higher oxidative stress status, were found in patients with dementia compared to healthy subjects., Conclusion: We established and validated a robust LC-MS/MS method to simultaneously measure 8-oxoGuo, 8-oxodG, and NMN in serum and urine., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: It is currently unclear for the necessary of pre-heating urine samples for the accurate determination of 8-oxo-7,8-dihydroguanosine (8-oxoG) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG). Thus, we conducted this study to evaluate the effect of pre-heat (i.e., to 37°C) on the accurate measurement of 8-oxoG and 8-oxodG in frozen urine samples., Methods: Random urine samples from six healthy volunteers, six patients with renal dysfunction, and six patients with systematic diseases such as diabetes were collected, split, and stored at -80°C for up to 1 month. The frozen samples were thawed at room temperature (RT) or 37°C for different time, 10-fold diluted with ddH2O containing 1% formic acid, and determined by self-established LC-MS/MS method coupled with an ACQUITY™ Primer HSS T3 column., Results: Thawing the samples at RT for 30 or 120 min, or at 37°C for 15 or 90 min did not affect the determination of 8-oxoG and 8-oxodG in urine samples. Moreover, no significant difference between thawing the urine samples at RT and 37°C was found after storing at -80°C for 1-3 months., Conclusion: It is not always necessary to pre-heat the frozen urine samples to release 8-oxoG and 8-oxodG from precipitates, which is associated with different pre-treatment and determination methods., (© 2022 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.)

Objectives: Measurement of the serum levels of vitamin B12 (VB12) is key for evaluating VB12 deficiency-dependent anemia. Immunoassay, the major method for determining VB12, tends to give false-normal results because of the presence of anti-intrinsic factor (IF-Ab) or other factors such as heterophilic antibodies et al. This study aimed to develop a liquid chromatography tandem mass spectrometry (LC-MS/MS) method that is helpful for distinguish false normal VB12 results measured by the immunoassay., Methods: Different forms of VB12 were derivatized into CN-B12, which was collected through solid-phase extraction and analyzed via LC-MS/MS. 236 serum samples were measured both by LC-MS/MS and immunoassay, results were compared, and the IF-Ab effect was evaluated., Results: The LC-MS/MS assay afforded a linear slope from 20 to 4,000 pmol/L for CN-B12. OH-VB12, methyl-VB12, and CoA-VB12 showed recovery within 89.3-109.5%. The intra-assay CV of VB12 was 2.6-4.1%, whereas the total CV was 9.3-9.8%. Passing-Bablok regression between LC-MS/MS and immunoassay results showed that the slope was 1.085 and the intercept was -15.691. The Bland-Altman plot showed that the mean difference and difference% were -34.6 pmol/L and 0.3%, respectively. Inter-rater agreement analysis showed that the linear weighted kappa value was 0.885, implying good agreement between the two methods. However, two samples were falsely elevated and one sample was falsely normal in the immunoassay compared with LC-MS/MS. The LC-MS/MS method helped in the distinction of false-normal VB12 results shown by the immunoassay., Conclusions: The VB12 LC-MS/MS method can be used as an arbiter of clinically discordant immunoassay results., (© 2022 Walter de Gruyter GmbH, Berlin/Boston.)

Background: Hemoglobin A 1c (HbA 1c ) is widely used to measure glycemic status and control in diabetes testing and treatment and is an important risk factor forcomplications of diabetes. Hemoglobin variants can interfere with the HbA 1c testing method by reducing the life span of erythrocytes or due to differences in glycation degrees. In this study, glycation levels of the HbA, HbG-Coushatta, and HbG-Taipei β-chains (βA, βG-Coushatta, and βG-Taipei, respectively) were examined., Methods: Blood samples from heterozygotic patients (HbG-Coushatta/HbA, HbG-Taipei/HbA) were analyzed. Glycation rateswere determined using high-performance liquid chromatography with tandem mass spectrometry. Ratios of glycated βG-Coushatta to glycated βA and glycated βG-Taipei to glycated βA were calculated by comparingareas under the curves from extracted ion chromatograms., Results: βG-Coushatta and βG-Taipei were 6.08 ± 1.38% and 5.95 ± 0.93% glycated (respectively), which were significantly higher than βA chains(4.55 ± 1.30% and 4.51 ± 0.91%, respectively; p = 0.000). The total glycation degree (α + β) in HbG-Coushatta and HbG-Taipei heterozygotes were estimated to be 9% and 8% higher than those of HbA homozygotes (P＜0.001), respectively., Conclusion: βG-Coushatta and βG-Taipei glycation degrees were significantly higher than βA, while the differences in total hemoglobin (α + β) were small and unlikely to impact the clinical interpretation of HbA 1c results., (Copyright © 2021. Published by Elsevier B.V.)

Background: Vitamins A and E play important roles in sustaining life activities and maintaining a good physical condition. However, most people, particularly the elderly, experience micronutrient deficiencies. This study aimed to establish reference intervals (RIs) for vitamins A and E in Chinese elderly people using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method., Methods: A total of 356 apparently healthy individuals aged ≥64 years who underwent health checkups were randomly selected for the study. Vitamin A and E levels were measured using LC-MS/MS. The effect of sex on vitamin A and E levels was evaluated, and RIs were established using a parametric method., Results: Females showed significantly higher levels of vitamin E than males (p < 0.05). However, no significant sex-specific difference was observed with vitamin A levels. The RI for vitamin A in the elderly was 0.283-0.730 mg/L. For vitamin E, the RIs were 4.39-15.63, 4.51-16.14, and 4.41-14.67 mg/L for the total, female, and male participants, respectively. In multiple linear regression, alanine aminotransferase, glutamyl transpeptidase, urea, glucose, and uric acid levels increased with increasing vitamin A levels (p < 0.05), and total cholesterol and low-density lipoprotein cholesterol levels increased with increasing vitamin E levels (p < 0.05). Direct bilirubin levels decreased with increasing vitamin E levels (p < 0.05)., Conclusions: This study established RIs for vitamins A and E in Chinese elderly individuals using an LC-MS/MS method. We also found that females had significantly higher vitamin E levels than males. The findings could provide a scientific basis for interpreting vitamin status in the elderly., (© 2021 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.)

Background This study aimed to quantify and compare serum aldosterone (sALD) levels through three different chemiluminescence immunoassays (CLIAs) and liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. Methods Serum samples from 221 patients with suspected primary aldosteronism (PA) were retrospectively included in this study conducted at the Peking Union Medical College Hospital from June to August in 2017. sALD levels were determined using the LC-MS/MS method and three different CLIA systems, viz., DiaSorin® XL, iSYS and Auto Lumo A2000. Pooled fresh serum samples were used for recalibration. Passing-Bablok regression analysis, correlation matrix, and Bland-Altman plots were used to evaluate the concurrence among ALD levels determined using the three CLIAs. Results Within-laboratory precision of the four assays ranged from 2.1% to 9.4%, except the coefficient variation (CV) of one of the CLIAs, which exceeded 20.0% for samples with low sALD levels. sALD levels determined using LC-MS/MS were significantly lower than those determined using the other three CLIAs (p < 0.0001). Spearman's correlation coefficient of the four assays ranged from 0.745 to 0.950 (p < 0.0001). The Bland-Altman plot showed that the average bias (%) for the three CLIAs and LC-MS/MS ranged from -69.3 to -49.2. After recalibration, this correlation did not improve among the assays. However, the bias and bias percentage at the medical decision level improved between LC-MS/MS and DiaSorin® XL/iSYS. Conclusions Significant inconsistencies between the results of CLIAs and LC-MS/MS indicate that different sALD measures cannot be used interchangeably.

Catecholamines [dopamine (DA), epinephrine (E), and norepinephrine (NE)] and their metabolites [metanephrine (MN), normetanephrine (NMN), and 3-methoxytyramine (3-MT)] are functionally important in humans. Their overexpression can indicate the presence of neuroendocrine tumors. Accurate and rapid quantitation of catecholamines and their metabolites may function in differential diagnosis of neuroendocrine tumors. Herein, we diluted 200 μL plasma using isotope labelled internal standards (IS), and extracted using solid phase extraction. The performance of isotope diluted liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) was evaluated and applied to quantify the level of catecholamines and metabolites in clinical samples from 73 apparently healthy adults. The total analysis time of the ID-LC-MS/MS method was 4 min. The improved method was highly sensitive, with a limit of quantification (LOQ) for MN, NMN, 3-MT, and E of 1 pg/mL, a LOQ for DA of 5 pg/mL, and for NE of 10 pg/mL. After correction using IS, no significant matrix effects were observed. Good reproducibility was obtained, with total CVs of 3.2-13.1% (DA), 4.8-10.0% (E), 6.2-6.9% (NE), 3.8-7.9% (MN), 4.1-8.8% (NMN), 3.4-8.9% (3-MT). Recoveries were in the range of 91.1-109.7% for the six analytes. Also, the mean concentration of catecholamines were as follows: MN, 22.9 ± 7.2 pg/mL; NMN, 41.4 ± 17.2 pg/mL; 3-MT, 2.34 ± 2.01 pg/mL; DA, 10.2 ± 4.6 pg/mL; E, 29.3 ± 14.2 pg/mL and NE 427.0 ± 190.6 pg/mL. A reliable ID-LC-MS/MS method for the determination of catecholamines and their metabolites using small volumes of plasma was verified. This method is rapid, simple, and may serve as an essential diagnostic tool for neuroendocrine tumors in clinical practice., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Accurate serum total thyroxine (TT4) measurement is important for thyroid disorder diagnosis and management. We compared the performance of six automated immunoassays with that of isotope-diluted liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) as the reference method. We also evaluated the correlation of thyroid stimulating hormone (TSH) with TT4 measured by ID-LC-MS/MS and immunoassays., Methods: Serum was collected from 156 patients between October 2015 and January 2016. TT4 was measured by immunoassays from Abbott (Architect), Siemens (ADVIA Centaur XP), Roche (E601), Beckman-Coulter (Dxi800), Autobio (Autolumo A2000), and Mindray (CL-1000i), and by ID-LC-MS/MS. Results were analyzed using Passing-Bablok regression and Bland-Altman plots. Minimum requirements based on biological variation were as follows: a mean bias of ≤4.5% and total imprecision (CV) of ≤3.7%., Results: All immunoassays showed a correlation >0.945 with ID-LC-MS/MS; however, the slope of the Passing-Bablok regression line varied from 0.886 (Mindray) to 1.23 (Siemens) and the intercept from -12.8 (Siemens) to 4.61 (Mindray). Only Autobio, Beckman-Coulter, and Roche included the value of one in the 95% confidence interval for slope. The mean bias ranged from -10.8% (Abbott) to 9.0% (Siemens), with the lowest value noted for Roche (3.5%) and the highest for Abbott (-10.8%). Only Abbott and Roche showed within-run and total CV ≤3.7%., Conclusions: Though all immunoassays correlated strongly with ID-LC-MS/MS, most did not meet the minimum clinical requirement. Laboratories and immunoassay manufacturers must be aware of these limitations., Competing Interests: No potential conflicts of interest relevant to this article were reported., (© The Korean Society for Laboratory Medicine.)

Aldosterone (ALD) measurement plays a critical role in screening and diagnosis of primary aldosteronism. A variety of liquid chromatography tandem mass spectrometry (LC-MS/MS) methods for serum ALD have been developed. However, the few LC-MS/MS methods for 24 h urinary ALD that have been reported require tedious sample pretreatment procedure. This study aimed to establish a novel method for quantification of ALD in urine by LC-MS/MS. Urine samples from study participants were hydrolyzed and subjected to anion exchange solid phase extraction (SPE) followed by the detection of eluates by the negative electro-spray ionization and multiple reaction monitor modes. The established method was compared with two CLIA methods and 114 participants (M:39, F:75) were recruited to study the distribution of urinary ALD in an apparently healthy population. The pretreatment time was reduced to 4 h and the total run time for each sample was 4.5 min. The linearity of the method was in the range of 2-2000 pg/mL (r > 0.999); within laboratory coefficient variation (CV) and repeatability were both <5% and recovery was within 100% ± 10%. The established method showed good consistency with two CLIA methods with the correlation coefficients of 0.993(DiaSorin) and 0.980(Auto), respectively. In the 114 apparent healthy individuals, urinary ALD was 0.74-17.09 μg/24 h (P2.5-P97.5). The method showed an excellent performance of the method, prompting us to conclude that this method may be adopted in the clinic for routine testing and analysis of ALD in urine samples., (Copyright © 2019. Published by Elsevier B.V.)

This study establishes and validates a rapid method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) without derivatization steps to simultaneously measure of 24,25(OH) 2 D 2 , 24,25(OH) 2 D 3 , 25OHD 2 , and 25OHD 3 , while efficiently separating the 3-epi analogs. Samples were prepared by precipitation and liquid-liquid extraction. The linearity, precision, accuracy, recovery and matrix effect of the method were thoroughly evaluated according to the Clinical & Laboratory Standards Institute guidelines. Additionally, the four vitamin D metabolites in the serum of 38 apparently healthy Chinese volunteers were evaluated. The total analysis time was 8.0 min, with efficient separation of 3-epi 24,25(OH) 2 D 3 and 3-epi 25OHD 3 , without interference from isomers such as 23,25(OH) 2 D 3 or 1,25(OH) 2 D 2 , 1,25(OH) 2 D 3 . Good reproducibility was obtained for all four metabolites with within-run coefficient variations (CVs) of 4.07%-6.55%, 4.26%-7.84%, 2.46%-7.21%, and 4.90%-6.87% for 25OHD 3 , 25OHD 2 , 24,25(OH) 2 D 3 , and 24,25(OH) 2 D 2 , respectively, and the total CVs were 4.29%-6.64%, 6.14%-7.84%, 4.33%-7.21%, 5.82%-9.90%, respectively. The limit of quantification was 0.625 ng/mL for 25OHD 3 and 25OHD 2 , and 0.5 ng/mL for 24,25(OH) 2 D 3 and 24,25(OH) 2 D 2 . The relative bias of the LC-MS/MS method compared to the certified results of SRM 972a for 25OHD 3 , 25OHD 2 and 24,25(OH) 2 D 3 was -2.21% to 1.01%, 3.38% to 6.73%, and -7.72% to -3.9%, respectively. The mean±SD values for 25OHD, 24,25(OH) 2 D and 25OHD/24,25(OH) 2 D in the volunteers were 13.5±4.4 ng/mL(range:7.6-27.5 ng/mL), 0.84±0.42 ng/mL (range:0.26-2.1 ng/mL), and 18±7(range:8-37), respectively. Thus, a simple, precise LC-MS/MS method for appropriate retention and separation of vitamin D metabolites and their epi analogs was developed., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Objectives: To develop a rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method with ability to separate 3-epi 25OHD3 (EPI-LC-MS/MS) from 25OHD3, and evaluate the effects of 3-epi 25OHD3 on routine LC-MS/MS that cannot separate 3-epi 25OHD3 (NEPI-LC-MS/MS)., Design and Methods: Performance of the newly built EPI-LC-MS/MS was validated, and 982 samples were analyzed and compared by the two methods., Results: Both methods showed a linearity coefficient correlation exceeding 0.999 in the 6.25-500nmol/L range for 25OHD2 and 25OHD3. Moreover, they showed a between run coefficient variation (CV) and total CV of < 5% for 25OHD2 and 25OHD3. The results of the accuracy test showed that the bias was below 6.19% in the absence of 3-epi 25OHD3. Comparison of the 25OHD results obtained by the two methods for 982 patients (age 1-100years) revealed excellent clinical agreement (Cohen's kappa=0.875) and correlation (R2=0.973). Among the 982patients, only 73patients had 3-epi 25OHD3 (>6.25nmol/L); out of these 73patients, the 3-epi 25OHD3 level in 58patients was between 6.25 and 12.5nmol/L. In patients with <375nmol/L 25OHD (25OHD2+25OHD3), only 8 had 3-epi 25OHD3 levels exceeding 12.5nmol/L (range: 13.3 - 27.5nmol/L). Among samples containing 3-epi 25OHD3, only three were separated into different 25OHD-deficiency groups using the above methods., Conclusion: A rapid and precise EPI-LC-MS/MS method for measuring 25OHD with efficient separation of 3-epi 25OHD3 was developed. Our results showed that 3-epi 25OHD3 had little effect on the routinely used NEPI-LC-MS/MS., (Copyright © 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Vitamin D plays important roles in skeletal metabolism and many other diseases, including chronic renal failure, hypoparathyroidism, sarcoidosis and rickets. 1α,25-dihydroxy vitamin D (1α,25(OH)2D), the active form of vitamin D, exhibits an extremely low serum concentration, which makes its quantification very challenging. High performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) is considered to be the "gold standard" for the determination of these chemicals, which are found in low concentrations in the serum, but conventionally, it needs tedious sample pretreatment procedures, such as solid phase extraction and derivatization. Herein, we describe a simple and rapid HPLC-MS/MS method for the simultaneous quantification of 1α,25-dihydroxy vitamin D3 (1α,25(OH)2D3) and 1α,25-dihydroxy vitamin D2 (1α,25(OH)2D2). The analytes were extracted from the matrix by liquid-liquid extraction, centrifuged to dryness and reconstituted with 75% methanol. Lithium acetate was employed to improve the ionization efficiency of 1α,25(OH)2D. The assay was sensitive with a low limit of quantitation of 10.0pg/mL for both 1α,25(OH)2D3 and 1α,25(OH)2D2 using a 0.5mL sample aliquot. Linearity was obtained over the range of 10.0pg/mL to 500pg/mL. Both the inter-assay and intra-assay precisions were <15%, and the analytical recoveries were within 100±5%. The performance evaluation of this assay demonstrated that it was a practical, sensitive and specific method for measuring the serum 1α,25(OH)2D3 and 1α,25(OH)2D2 concentrations., (Copyright © 2016 Elsevier B.V. All rights reserved.)

We described a rapid and precise method for simultaneous quantification of eleven fatty acids in human serum cholesteryl esters (CEFAs) by liquid chromatography and tandem mass spectrometry (LC-MS/MS). After extraction of serum lipids with isopropanol, CEFAs were separated on reversed phase liquid chromatography and detected by mass spectrometry in positive ion mode with multiple reaction monitor. Individual CEFA was quantified by peak area normalization method and expressed as molar percent of total CEFAs. The run time was less than 5 min and detection limits were from 0.31 to 14.50 × 10(-5)mmol/L. Recoveries of the CEFAs ranged from 91.85% to 104.83% with a mean of 99.12%. The intra and total CVs for the measurement of CEFAs were 0.87-7.70% and 1.02-7.65%, respectively. This LC-MS/MS method required no internal standards, eliminated natural isotope interferences, and provided reproducible and reliable results for 11 major CEFAs in human serum. This method can be used in monitoring and evaluating dietary fatty acid intake. Additional studies are needed to evaluate the associations between serum CEFAs and cardiovascular disease risk factors., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Serum branched-chain and aromatic amino acids (BCAAs and AAAs) have emerged as predictors for the future development of diabetes and may aid in diabetes risk assessment. However, the current methods for the analysis of such amino acids in biological samples are time consuming., Methods: An isotope dilution liquid chromatography tandem mass spectrometry (ID-LC/MS/MS) method for serum BCAAs and AAAs was developed. The serum was mixed with isotope-labeled BCAA and AAA internal standards and the amino acids were extracted with acetonitrile, followed by analysis using LC/MS/MS. The LC separation was performed on a reversed-phase C18 column, and the MS/MS detection was performed via the positive electronic spray ionization in multiple reaction monitoring mode., Results: Specific analysis of the amino acids was achieved within 2 min. Intra-run and total CVs for the amino acids were less than 2% and 4%, respectively, and the analytical recoveries ranged from 99.6 to 103.6%., Conclusion: A rapid and precise method for the measurement of serum BCAAs and AAAs was developed and may serve as a quick tool for screening serum BCAAs and AAAs in studies assessing diabetes risk.

Background: This study aimed to quantify and compare serum aldosterone (sALD) levels through three different chemiluminescence immunoassays (CLIAs) and liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. Methods: Serum samples from 221 patients with suspected primary aldosteronism (PA) were retrospectively included in this study conducted at the Peking Union Medical College Hospital from June to August in 2017. sALD levels were determined using the LC-MS/MS method and three different CLIA systems, viz., DiaSorin® XL, iSYS and Auto Lumo A2000. Pooled fresh serum samples were used for recalibration. Passing-Bablok regression analysis, correlation matrix, and Bland-Altman plots were used to evaluate the concurrence among ALD levels determined using the three CLIAs. Results: Within-laboratory precision of the four assays ranged from 2.1% to 9.4%, except the coefficient variation (CV) of one of the CLIAs, which exceeded 20.0% for samples with low sALD levels. sALD levels determined using LC-MS/MS were significantly lower than those determined using the other three CLIAs (p < 0.0001). Spearman's correlation coefficient of the four assays ranged from 0.745 to 0.950 (p < 0.0001). The Bland-Altman plot showed that the average bias (%) for the three CLIAs and LC-MS/MS ranged from −69.3 to −49.2. After recalibration, this correlation did not improve among the assays. However, the bias and bias percentage at the medical decision level improved between LC-MS/MS and DiaSorin® XL/iSYS. Conclusions: Significant inconsistencies between the results of CLIAs and LC-MS/MS indicate that different sALD measures cannot be used interchangeably. [ABSTRACT FROM AUTHOR]

Very-long-chain fatty acids (VLCFAs), including hexacosanoic, tetracosanoic, and docosanoic acids, are peroxisomal disease markers, whose abnormal accumulation warrants prompt detection for timely, effective treatment. This study aimed to establish and validate a robust liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method to simultaneously quantify VLCFAs and provide reference intervals among Chinese individuals, quantify VLCFAs in pregnancy, and explore potential associations between plasma and amniotic fluid. Analytes were extracted via water-bath incubation with HCl and liquid-liquid extraction. Method linearity, limit of detection/quantitation, precision, carryover, and recovery were evaluated according to Clinical and Laboratory Standard Institute (CLSI) guidelines. VLCFAs showed good reproducibility based on low within-run coefficient variations (CVs) and total CVs, and correlation coefficients of linearity were > 0.99. The reference interval of C22:0, C24:0, and C26:0 were 32.0–73.4 μmol/L, 30.3–72.0 μmol/L, and 0.20–0.71 μmol/L, respectively; C24:0/C22:0 and C26:0/C22:0 ratios were 0.75–1.28 and 0.005–0.0139, respectively. Plasma and amniotic fluid of the same pregnant women displayed no significant correlation in the second trimester. This study presents the simple, efficient, accurate, and robust LC-MS/MS method to simultaneously detect C22:0, C24:0, and C26:0 without derivatization; it can be used to establish reference intervals among Chinese individuals and has diagnostic and other clinical applications. • LC-MS/MS to detect very-long-chain fatty acids (VLCFAs) was successful in China. • Reference intervals of VLCFAs were established for the Chinese population. • VLCFAs were compared between plasma and amniotic fluid in late pregnancy. • Simultaneous VLCFAs detection can improve the diagnosis of peroxisomal disorders. [ABSTRACT FROM AUTHOR]

• 24,25(OH) 2 D and the 25OHD/24,25(OH) 2 D ratio were firstly evaluated in a Chinese population. • Sources of variations in 24,25(OH) 2 D and the 25OHD/24,25(OH) 2 D ratio were studied. • 24,25(OH) 2 D and 25OHD showed significant sex differences, but their ratio did not. 24,25(OH) 2 D and the 25OHD/24,25(OH) 2 D ratio have recently been shown to be useful for screening for hypercalcemia caused by CYP24A1 mutations. However, no population-based data on 24,25(OH) 2 D have been published for Chinese populations, and few studies evaluated the sources of variation on 24,25(OH) 2 D levels and the 25OHD/24,25(OH) 2 D ratio. Hence, in 2018, we enrolled 1211 apparently healthy adults without systematic diseases from six representative cities in China (Beijing, Dongying, Guiyang, Urumqi, Shenzhen, and Qiqihar). 24,25(OH) 2 D and 25OHD levels were measured using isotope dilution liquid chromatography tandem mass spectrometry. Multiple regression analysis showed that sex contributed the most to variations in 24,25(OH) 2 D, 25OHD, and 25OHD/24,25(OH) 2 D (rp= -0.255, -0.253, and 0.141, respectively), and age also contributed to variations in 25OHD but not 24,25(OH) 2 D or 25OHD/24,25(OH) 2 D. Men had significantly higher 24,25(OH) 2 D and 25OHD levels than women, but had significantly lower 25OHD/24,25(OH) 2 D values. Individuals from Dongying had the highest 24,25(OH) 2 D and 25OHD values, whereas individuals from Urumqi had the lowest values. The median(2.5–97.5%) values for 24,25(OH) 2 D, 25OHD, and 25OHD/24,25(OH) 2 D were 1.2(0.36–2.65) ng/mL, 18.9(8.6–32.5) ng/mL, and 16.0 (9.8–30.8), respectively. 24,25(OH) 2 D was significantly correlated with 25OHD (r = 0.838, p < 0.001), and 25OHD/24,25(OH) 2 D was significantly negatively correlated with 24,25(OH) 2 D (r = -0.758, P < 0.001) and 25OHD (r = -0.310, P < 0.001). In conclusion, in this nationwide, multicenter, cross-sectional study, we evaluated the levels of 24,25(OH) 2 D and the 25OHD/24,25(OH) 2 D ratio in the Chinese population. Sex contributed the most to variations in 24,25(OH) 2 D, 25OHD, and 25OHD/24,25(OH) 2 D. [ABSTRACT FROM AUTHOR]