Vitamin D plays important roles in bone health and a variety of other pathophysiological conditions. 1α,25-Dihydroxyvitamin D is the active form of vitamin D. Quantification of serum 1α,25-dihydroxyvitamin D is useful for evaluation of several diseases including chronic renal failure, hypoparathyroidism, sarcoidosis, and rickets. Measurement of 1α,25-dihydroxyvitamin D is very challenging due to its low circulating concentration and presence of interfering substances in serum. In this report, a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for quantifying serum 1α,25-dihydroxyvitamin D is described. Lithium adducts of 1α,25-dihydroxyvitamin D were formed prior to mass spectrometry analysis to improve ionization efficiency. We tested a number of different sample preparation procedures and found that immunoaffinity extraction was the method of choice because it completely removed isobaric interferences and matrix effects present in patient serum. Extraction efficiency, expressed as absolute recovery, was greater than 60% in both patient serum and charcoal-stripped serum. This method was linear from 3.4 to 206.2 pg/mL for 1α,25-dihydroxyvitamin D(3) and 3.9 to 212.6 pg/mL for 1α,25-dihydroxyvitamin D(2) with an accuracy of 89.8-98.4% and 97.5-115.7%, respectively. Inter-assay and intra-assay coefficients of variance (CVs) for both analytes at two different concentration levels ranged from 2.5-7.0%. Comparison with a radioimmunoassay for measuring total 1α,25-dihydroxyvitamin D concentration using 40 patient samples showed a Deming regression slope of 0.751, a y-intercept of 0.84 pg/mL, an r value of 0.7909, and a mean percentage difference of -27.1%. Comparison with a reference LC/MS/MS method (n = 20) showed a Deming regression slope of 1.020, a y-intercept of 1.32 pg/mL, an r value of 0.9797, and a mean percentage difference of -2.9%. In conclusion, usage of immunoaffinity extraction enabled a sensitive LC/MS/MS method for quantification of 1α,25-dihydroxyvitamin D in serum.

Oral fluid has been gaining more acceptance as the alternative matrix for forensic toxicology. Currently, Δ(9)-tetrahydrocannabinol (THC) is used as the primary target for detecting cannabis use in oral fluid. Meanwhile, THC carboxylic acid (THCA) in oral fluid is reported as a more reliable marker for cannabis abuse as its presence does not come from passive exposure. An analytical method for simultaneous quantitation of THC and THCA will be efficient for toxicology laboratories. THCA quantitation is challenging due to its very low concentration in oral fluid. Recently reported liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based methods achieved sufficient sensitivity but involved complex sample preparation procedures. We aimed to develop a sensitive LC-MS/MS method for simultaneous quantitation of THC and THCA in oral fluid with low-flow liquid chromatography and a Q Exactive mass spectrometer, using offline sample preparation of oral fluid followed by microflow LC with online sample cleanup. The total runtime of the method was 12.5 min. The method had a lower limit of quantitation of 7.5 pg/mL and was linear from 7.5 to 300 pg/mL for THCA. The intra- and interbatch precision of the method ranged from 3.3% to 9.3% for THC and THCA.

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Liquid chromatography-tandem mass spectrometry has become the preferred technology to measure unconjugated metanephrine and normetanephrine in plasma because of its high sensitivity and specificity over immunoassay and gas chromatography-mass spectrometry. In our earlier study, plasma metanephrines were extracted with offline ion-pairing solid-phase extraction and quantified by liquid chromatography-tandem mass spectrometry with porous graphitic carbon column based chromatography. In this study, we aim to automate the sample preparation with turbulent flow online extraction technology and maintain or improve the analytical performance previously achieved from the offline approach. The online extraction was done with a mixed-mode cation exchange turbulent flow chromatography column assisted with ion-pairing reagent and porous graphitic column was used for chromatographic separation. The total online extraction and analytical LC runtime was 12 min. This method was linear from 6.3 to 455.4 pg/mL for metanephrine; 12.6 to 954.5 pg/mL for normetanephrine with an accuracy of 80.6% to 93.5% and 80.9% to 101.7%, respectively. The lower limit of quantitation was 6.3 pg/mL for metanephrine and 12.6 pg/mL for normetanephrine. Inter-assay and intra-assay precision for metanephrine and normetanephrine at low and high concentration levels ranged from 2.0% to 10.5%. In conclusion, we have developed a fast and sensitive automated online turbulent flow extraction method for the quantitative analysis of plasma metanephrines. Ion-pairing reagent was necessary for the success of this method.

Urine and plasma specimens fortified with 82 drugs and metabolites were prepared and analyzed by liquid chromatography quadrupole time-of-flight mass spectrometry (QTOF) instrumentation from three different vendors using the instrument manufacturers' methods and workflows for drug screening. No prior knowledge about the compounds included or their concentrations were provided. Samples were prepared and sent for analysis on a TripleTOF(®) 5600 system, a 6530 QTOF and a Xevo(®) G2-S QTof. All three platforms performed well with >90% of compounds detected in one set of spiked plasma samples, and 79-88% for a second set of spiked plasma and two sets of spiked urine samples.