Reference intervals for LC-MS /MS measurements of plasma renin activity, aldosterone, angiotensin II, and 24-hour urinary aldosterone in Northern Chinese Han population.

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.

An automated magnetic bead extraction method for measuring plasma metanephrines and 3-methoxytyramine using liquid chromatography tandem mass spectrometry.

Liquid chromatography tandem mass spectrometry (LC-MS/MS) is used routinely in clinical diagnostics; however, automating the sample pretreatment is challenging. We established and evaluated an automated method based on the magnetic bead extraction principle (MBE) to measure normetanephrine (NMN), metanephrine (MN), and 3-methoxytyramine (3-MT). The target analytes were extracted, purified, and concentrated using different solvents and chemical bond-modified magnetic beads transferred via a magnetic bar. The linearity, recovery, matrix effect, and precision of MBE were evaluated thoroughly, and compared with traditional solid-phase extraction (SPE) using 131 plasma samples. The chromatography peaks of metanephrines and 3-MT, extracted via MBE, are symmetrical, without interfering peaks. The linearity was excellent with correlation coefficient (r) > 0.99. The MBE exhibited good reproducibility with within-run coefficient variations (CVs) of 1.96-2.00%, 4.06-5.75%, and 3.89-4.90% for MN, NMN, and 3-MT, respectively. The total CVs for MN, NMN, and 3-MT were 1.96-2.80%, 5.12-5.75%, and 5.44-6.27%, respectively. The relative recoveries for MN, NMN, and 3-MT varied between 93.5 and 107.4%, whereas their biases were all within 10%. The results for MN, NMN, and 3-MT extracted via MBE compared with SPE exhibited excellent correlation, with r > 0.99; the mean bias% for MN, NMN, and 3-MT were small (-2.9%, -3.2%, and -3.2%, respectively). In conclusion, the automated MBE method for measuring plasma metanephrines and 3-MT can be applied in future routine clinical diagnostics, and the MBE principle may indicate a new era for LC-MS/MS in clinical application.

Analytical and Clinical Performance of a Liquid Chromatography-Tandem Mass Spectrometry Method for Measuring Gastrin Subtypes G34 and G17 in Serum.

BACKGROUND: Two major forms of gastrin, gastrin-17 (G17) and gastrin-34 (G34), exist in blood. However, conventional immunoassay methods can only quantify total gastrin or G17 alone. Here, we aimed to establish a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify G17 and G34 simultaneously. METHODS: Serum samples were prepared by anion-exchange solid-phase extraction. The analytical performance of the LC-MS/MS method was validated and the method was compared to chemiluminescence immunoassay (CLIA) and radioimmunoassay (RIA). The G17 and G34 concentrations in 245 serum samples from healthy controls, individuals with gastrinoma, and individuals with other diseases were analyzed. RESULTS: The total runtime of the LC-MS/MS method was 6 min. No substantial matrix effect was observed with internal standard correction. The intraassay coefficients of variation (CVs) for G17 and G34 were 4.0%-14.2% and 4.4%-10.4%, respectively, and total CVs were 5.2%-14.1% and 4.6%-12.4%, respectively. The correlation coefficient between LC-MS/MS and CLIA was 0.87, and between LC-MS/MS and RIA was 0.84. The G17+G34 concentrations for 87.5% of individuals with gastrinoma were higher than the 95th percentile of healthy controls (18.1 pg/mL), whereas the concentrations for individuals with other diseases and gastrinoma overlapped. Based on the Youden indices calculated for G17+G34, G34, and G17, the most specific biomarker was G17 (96.9% clinical specificity at 209.8 pg/mL) for gastrinoma. CONCLUSIONS: This method should aid in the diagnosis of diseases associated with increased gastrin concentrations.

Plasma or serum, which is the better choice for the measurement of metanephrines?

Measurement of metanephrines (MNs: metanephrine [MN] and normetanephrine [NMN]) is recommended for the initial biochemical diagnosis of pheochromocytoma and paraganglioma. Despite some drawbacks, plasma is commonly used for sampling. Here, we determined the feasibility of using serum, as an alternative to plasma, by comparing MNs in plasma and serum and evaluating the stability of MNs in serum. MNs obtained from serum, EDTA plasma, and heparin plasma were measured using LC-MS/MS immediately or after storage at 4 °C for 24 h, 72 h, and 7 days, and at -80 °C for 7 days, after sample collection. The differences between sample stability at given time points were compared using one-way ANOVA and Students' paired t-test, and the mean percent deviation was compared with total change limit (TCL). No significant difference was observed in MN and NMN between serum and EDTA plasma, and the mean percent deviation of the results obtained from serum compared to that from EDTA plasma was within the TCL. However, the difference of MN between EDTA plasma and heparin plasma exceeded the TCL. Both MNs in EDTA plasma and heparin plasma showed a significant decreasing trend at 4 °C with time (p < .01), while those in serum were relatively stable, with the mean percent deviation not exceeding the TCL at any time point or temperature. In conclusion, MNs measurement did not significantly differ between EDTA plasma and serum when measured immediately after collection, and MNs in serum were more stable than that in plasma.

Validation of an improved liquid chromatography tandem mass spectrometry method for rapid and simultaneous analysis of plasma catecholamine and their metabolites.

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 ±â€¯7.2 pg/mL; NMN, 41.4 ±â€¯17.2 pg/mL; 3-MT, 2.34 ±â€¯2.01 pg/mL; DA, 10.2 ±â€¯4.6 pg/mL; E, 29.3 ±â€¯14.2 pg/mL and NE 427.0 ±â€¯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.