Multiple reaction monitoring with multistage fragmentation (MRM3) detection enhances selectivity for LC-MS/MS analysis of plasma free metanephrines.

BACKGROUND: LC-MS/MS with multiple reaction monitoring (MRM) is a powerful tool for quantifying target analytes in complex matrices. However, the technique lacks selectivity when plasma free metanephrines are measured. We propose the use of multistage fragmentation (MRM(3)) to improve the analytical selectivity of plasma free metanephrine measurement. METHODS: Metanephrines were extracted from plasma with weak cation exchange solid-phase extraction before separation by hydrophilic interaction liquid chromatography. We quantified normetanephrine and metanephrine by either MRM or MRM(3) transitions m/z 166→134→79 and m/z 180→149→121, respectively. RESULTS: Over a 6-month period, approximately 1% (n = 21) of patient samples showed uncharacterized coeluting substances that interfered with the routine assay, resulting in an inability to report results. Quantification with MRM(3) removed these interferences and enabled measurement of the target compounds. For patient samples unaffected by interferences, Deming regression analysis demonstrated a correlation between MRM(3) and MRM methods of y = 1.00x - 0.00 nmol/L for normetanephrine and y = 0.99x + 0.03 nmol/L for metanephrine. Between the MRM(3) method and the median of all LC-MS/MS laboratories enrolled in a quality assurance program, the correlations were y = 0.97x + 0.03 nmol/L for normetanephrine and y = 1.03x - 0.04 nmol/L for metanephrine. Imprecision for the MRM(3) method was 6.2%-7.0% for normetanephrine and 6.1%-9.9% for metanephrine (n = 10). The lower limits of quantification for the MRM(3) method were 0.20 nmol/L for normetanephrine and 0.16 nmol/L for metanephrine. CONCLUSIONS: The use of MRM(3) technology improves the analytical selectivity of plasma free metanephrine quantification by LC-MS/MS while demonstrating sufficient analytical sensitivity and imprecision.

Reporting Thyroid Function Tests in Pregnancy.

While there is agreement that overt maternal hypothyroidism (serum thyroid stimulating hormone (TSH) >10 mIU/L) should be treated immediately, the evidence is mixed regarding the harm associated with subclinical hypothyroidism and the benefits of thyroxine replacement. The diagnosis of subclinical hypothyroidism rests on the recognition of an increased serum concentration of TSH which may be affected by many factors including gestational age, analytical method, the antibody status of the mother, ethnicity, iodine nutrition and even the time of day when the blood is collected. The 97.5(th) percentile of TSH at the end of the first trimester is commonly used as the upper boundary of normal in early pregnancy with a default value of 2.5 mIU/L specified in a number of recent clinical guidelines. There have now been numerous papers showing that a more realistic figure is between 3.0 and 4.0 mIU/L depending on the analytical method that is used. There are suggestions that ethnicity may also have a significant effect on TSH and FT4 reference limits in pregnancy.