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Background The measurement of plasma concentrations of retinol binding protein is a component of nutritional assessment in neonatal intensive care. However, serial testing in newborns is hampered by the limited amount of blood that can be sampled. Limitations are most severe with preterm infants, for whom close monitoring may be most important. Methods We developed an assay to quantify retinol binding protein using trypsin digestion and liquid chromatography-tandem mass spectrometry, which requires a serum or plasma volume of 5 µl. Additionally, we validated the method according to current recommendations and performed comparison with a standard nephelometry platform in clinical use. Results The assay demonstrated linearity from below 1 mg/dL (0.48 µM) to more than 20 mg/dL (9.7 µM), and an imprecision of 11.8% at 0.43 mg/dL (0.21 µM). The distribution of results observed with the new method was different when compared with nephelometry. Conclusion Liquid chromatography-tandem mass spectrometry facilitated testing a smaller sample volume, thereby increasing the ability to monitor key nutritional markers in premature infants. The differences in results compared with a commercially-available nephelometric assay revealed questionable results for lower concentrations by immunoassay.

BACKGROUND: Serum 25-hydroxyvitamin D [25(OH)D] concentration is an indicator of vitamin D exposure, but it is also influenced by clinical characteristics that affect 25(OH)D production and clearance. Vitamin D is the precursor to 25(OH)D but is analytically challenging to measure in biological specimens. OBJECTIVES: We aimed to develop and validate a liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for quantification of vitamins D(3) and D(2) in serum and to explore the potential of circulating vitamin D as a biomarker of exposure in supplementation trials. METHODS: The method was validated using guideline C62-A from the Clinical and Laboratory Standards Institute and was applied in 2 pilot clinical trials of oral vitamin D(3) supplementation. Pilot study 1 included 22 adults randomly assigned to placebo or 2000 IU/d. Blood was collected at baseline, 1, 3, 6, and 12 mo. Pilot study 2 included 15 adults randomly assigned to 2000 or 4000 IU/d. Blood and subcutaneous (SUBQ) adipose tissue were collected at baseline and 3 mo. RESULTS: In study 1, mean change (baseline to 3 mo) in serum vitamin D(3) was −0.1 ng/mL in the placebo group and 6.8 ng/mL in the 2000 IU/d group (absolute difference: 6.9; 95% CI: 4.5, 9.3 ng/mL). In study 2, mean change (baseline to 3 mo) in serum vitamin D(3) was 10.4 ng/mL in the 2000 IU/d group and 22.2 ng/mL in the 4000 IU/d group (fold difference: 2.15; 95% CI: 1.40, 3.37). Serum and adipose tissue vitamin D(3) concentrations were correlated, and the dose-response of vitamin D(3) in adipose mirrored that in serum. CONCLUSIONS: We validated a sensitive, robust, and high-throughput LC-MS/MS method to quantify vitamins D(3) and D(2) in serum. Serum and SUBQ adipose tissue vitamin D(3) concentrations increased proportionally to dose with 3 mo of daily supplementation. These trials were registered at clinicaltrials.gov as NCT00552409 (pilot study 1) and NCT01477034 (pilot study 2).

Six laboratories associated with the Vitamin D Standardization Program (VDSP) participated in an interlaboratory comparison of LC with tandem MS (MS/MS) methods for the determination of 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] in human serum. The laboratories analyzed two different serum-based Standard Reference Materials (SRMs) intended for use in the determination of 25-hydroxyvitamin D and 30 samples from the Vitamin D External Quality Assessment Scheme (DEQAS). All laboratorymethods for 24,25(OH)2D3 were based on isotope dilution LC-MS/MS; three of the methods used derivatization of the vitamin D metabolites before LC-MS/MS. Laboratory results were compared to the National Institute of Standards and Technology (NIST) results, which were obtained using their newly developed candidate reference measurement procedure for 24,25(OH)2D3. Laboratory results for the SRM samples varied in comparability to the NIST results, with one laboratory in excellent agreement (−1.6% mean bias), three laboratories at 10–15% mean bias, and the remaining laboratory at 36% mean bias. For the 30 DEQAS samples, the mean bias for the five laboratories ranged from 6 to 15%; however, the SD of the bias ranged from 8 to 29%. As a result of this intercomparison study, one laboratory discovered and corrected a methodcalculation error and another laboratory modified and improved their LC-MS/MS method.

A 58-year-old woman with debilitating ankylosing spondylitis who was born to consanguineous parents was found to have an apparent severe vitamin D deficiency that did not respond to supplementation. Liquid chromatography-tandem mass spectrometry showed the absence of circulating vitamin D-binding protein, and chromosomal microarray confirmed a homozygous deletion of the group-specific component (GC) gene that encodes the protein. Congenital absence of vitamin D-binding protein resulted in normocalcemia and a relatively mild disruption of bone metabolism, in this case complicated by severe autoimmune disease. (Funded by the National Institutes of Health and the University of Washington.).

BACKGROUNDQuantifying the variability of biomarkers is important, as high within-person variability can lead to misclassification of individuals. Short-term variability of important markers of vitamin D metabolism is relatively unknown.METHODSA repeatability study was conducted in 160 Atherosclerosis Risk in Communities study participants (60% female, 28% black, mean age 76 years). Fasting serum was drawn at 2 time points, a median of 6 (range 3–13) weeks apart. Vitamin D binding protein (VDBP) and 25-hydroxyvitamin D [25(OH)D] were measured by LC-MS, fibroblast growth factor (FGF23) and parathyroid hormone (PTH) by enzyme-linked immunoassay, and calcium and phosphorus by Roche Cobas 6000. Free and bioavailable 25(OH)D were calculated. We calculated the within-person CV (CVW), intraclass correlation coefficient (ICC), Spearman rank correlation coefficient (r), and percent reclassified.RESULTSThe CVW was lowest for calcium (2.0%), albumin (3.6%), 25(OH)D (6.9%), VDBP (7.0%) and phosphorus (7.6%); intermediate for free 25(OH)D (9.0%) and bioavailable 25(OH)D (9.9%); and highest for PTH (16.7%) and FGF23 (17.8%). Reclassification was highest for PTH, VDBP, and phosphorus (all 7.5%). The ICC and r were highest (≥0.80) for 25(OH)D, free 25(OH)D, bioavailable 25(OH)D and PTH, but somewhat lower (approximately 0.60–0.75) for the other biomarkers.CONCLUSIONSSix-week short-term variability, as assessed by CVW, was quite low for VDBP, calcium and phosphorus, but fairly high for FGF23 and PTH. As such, multiple measurements of FGF23 and PTH may be needed to minimize misclassification. These results provide insight into the extent of potential misclassification of vitamin D markers in research and clinical settings.

The implementation of mass spectrometry to measure serum 25-hydroxyvitamin D [25(OH)D] concentrations has led to concerns regarding the measurement and reporting of the C3-epimer of 25-hydroxyvitamin D(3) [3-epi-25(OH)D(3)], for which there is a near-total lack of data regarding its clinical significance.We developed a chromatographic method to resolve (90%) 3-epi-25(OH)D(3) from 25(OH)D(3) using a pentafluorophenyl propyl chromatographic column. Using LC-MS/MS, we determined the serum concentrations of 25(OH)D(3) and 3-epi-25(OH)D(3) in 626 patients aged 3 days to 94 years undergoing routine vitamin D testing.Comparison between DiaSorin RIA and the new LC-MS/MS method for total 25(OH)D had acceptable agreement. Our data indicate an increase in 25(OH)D(3) rather than a reduction in epimer concentration. An average of 3.3 ng/ml of 3-epi-25(OH)D(3) was detected in adolescents and adults. Inclusion of 3-epi-25(OH)D(3) in the total 25(OH)D(3) concentration resulted in 9% (1 year) and 3% (1 to 94 years) potential misclassification of patients as vitamin D sufficient.The new LC-MS/MS method is capable of chromatographically separating 25(OH)D(3) and 3-epi-25(OH)D(3). It was used to confirm that the contribution of 3-epi-25OHD(3) to total 25OHD(3) concentrations decreases with age in infants and is detectable in adults.

BACKGROUND 1α,25-dihydroxy vitamin D [1,25(OH)2D] is the active metabolite of vitamin D. Antibody-based detection methods lack specificity, but when combined with isotope dilution/ultra-performance liquid chromatography (UPLC)–tandem mass spectrometry, immunoextraction provides an attractive method for 1,25(OH)2D. We developed a method for simultaneous quantification of 1,25(OH)2D2 and 1,25(OH)2D3 with a 4.6-min instrument cycle time. Results are available 36 h after sample preparation begins. METHODS Sample preparation consisted of protein precipitation, immunoextraction with solid-phase anti-1,25(OH)2D antibody, and derivatization with 4-phenyl-1,2,4-triazoline-3,5-dione. Analytes were resolved using reversed-phase UPLC and quantified using positive ion electrospray ionization–tandem mass spectrometry. We used hexadeuterated 1,25(OH)2D3 and 1,25(OH)2D2 as internal standards and performed method comparisons against the DiaSorin RIA and an LC-MS/MS method available at a reference laboratory. RESULTS 1,25(OH)2D3 intraassay and interassay imprecision was 5.6% and 8.0% (120 pmol/L) and 8.7% and 13% (48 pmol/L). Limits of detection and quantification were 1.5 pmol/L and 3.0 pmol/L, respectively. 1,25(OH)2D2 intraassay and interassay imprecision was 8.7% and 11% (186 pmol/L) and 11% and 13% (58 pmol/L). Limits of detection and quantification were both 1.5 pmol/L. Comparison with RIA had a proportional bias of 0.75, constant bias of −4.1, and Pearson correlation (r2) of 0.31. Comparison with a reference LC-MS/MS assay had a proportional bias of 0.89, constant bias of 3.7, and r2 of 0.88. CONCLUSIONS Protein precipitation with antibody-based extraction is effective for sample preparation before LC-MS/MS analysis of derivatized 1,25(OH)2D. This method appears to have improved specificity over a clinically used RIA with low imprecision and limits of detection.

Background: High-performance liquid chromatography–tandem mass spectrometric (LC-MS/MS)1 analysis of plasma free metanephrines is the most diagnostically sensitive and specific screening test for the diagnosis of pheochromocytoma. We sought to develop an in-house method for this expensive test Methods: We used off-line isopropanol protein precipitation of plasma to remove interfering substances before LC-MS/MS analysis. We compared the extraction efficiency and limits of quantification of protein precipitation to those of previously reported solid-phase techniques. Results: The new method had limits of quantification of 0.09 nmol/L and 0.17 nmol/L for metanephrine and normetanephrine, respectively. Method comparison with a previously described solid-phase extraction method revealed Deming regression slopes of 0.904 and 0.994, intercepts of 0.007 and 0.023, and SEs of the residuals (Sy|x) of 0.071 and 0.284 for metanephrine and normetanephrine, respectively. Extraction efficiency of isopropanol protein precipitation was 66% for metanephrine and 35% for normetanephrine, results that were superior to the efficiencies of 4% and 1% for our adapted solid-phase extraction method. No ion suppression was observed at the retention times for metanephrine and normetanephrine. Conclusions: Isopropanol protein precipitation is a novel and effective off-line sample preparation method for metanephrines that offers a less expensive alternative to on-line solid-phase extraction for low-volume testing and requires a sample volume of only 200 μL. The mass spectrometric analysis time is equivalent to that of solid-phase techniques.

BACKGROUND Vitamin D deficiency is associated with poor bone health and other adverse health outcomes; however, the associations are greatly attenuated in black vs white individuals. One possible explanation for this attenuation is different concentrations of bioavailable vitamin D metabolites in plasma, which are estimated with equations that include the total concentration of vitamin D binding globulin (VDBG) and haplotype-specific dissociation constants. METHODS We developed a method to quantify VDBG with LC-MS/MS that could also identify the haplotypes/isoforms of VDBG present. We validated the method according to recent recommendations for publications of biomarker studies. We determined serum VDBG concentrations in samples from the Atherosclerosis Risk in Communities cohort and compared the results with a widely used monoclonal immunoassay. RESULTS With 10 μL of serum or plasma, the lower limit of quantification for the assay ( CONCLUSIONS Validated mass spectrometric methods for the quantification of proteins in human samples can provide additional information beyond immunoassay. Counter to prior observations by immunoassay, VDBG concentrations did not vary by race.

Serum levels of 25-hydroxyvitamin D are important in establishing true vitamin D levels in humans. The purposes of this study were to develop a sensitive, specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for detection of 25-hydroxyvitamin D(2) and D(3) and establish reference intervals for these analytes. Chromatographic separation of 25(OH)D(2) and 25(OH)D(3) was achieved after adding deuterated Delta(9)-tetrahydrocannabinol (D(9)-THC-D(3)) and organic extraction. The 3 ions were ionized using positive electrospray ionization and detected in the multiple-reaction monitoring mode using mass (m)/charge (z) transitions of 318.15 > 196.20 (Delta(9)-THC-D(3)), 401.15 > 365.2 (25(OH)D(3)), and 413.15 > 355.20 (25(OH)D(2)). Reference interval study results were compared with current 25(OH)D recommendations. Elution of 25(OH)D(2), 25(OH)D(3), and Delta(9)-THC-D(3) was achieved after 3.0 minutes (total run time, 6.0 minutes). Within- and between-run coefficients of variation were less than 11%. Deming regression of radioimmunoassay and LC-MS/MS methods for total 25(OH)D levels yielded a slope of 0.97 (95% confidence interval, 0.88-1.05) and y-intercept of -1.74 ng/mL. Reference intervals were less than recommended levels (D(2), 0.0-12.1; D(3), 5.5-41.4; total vitamin D, 6.0-43.5 ng/mL [0-30, 14-103, 15-109 nmol/L, respectively]) with no statistically significant differences in race, age, or sex. This LC-MS/MS method provides a rapid, accurate, sensitive, and cost-effective alternative to other methods for detection of 25(OH)D(2) and 25(OH)D(3) at nanomolar concentrations.

Renal function generally is assessed by measurement of GFR and urinary albumin excretion. Other intrinsic kidney functions, such as proximal tubular secretion, typically are not quantified. Tubular secretion of solutes is more efficient than glomerular filtration and a major mechanism for renal drug elimination, suggesting important clinical consequences of secretion dysfunction. Measuring tubular secretion as an independent marker of kidney function may provide insight into kidney disease etiology and improve prediction of adverse outcomes. We estimated secretion function by measuring secreted solute (hippurate, cinnamoylglycine, p-cresol sulfate, and indoxyl sulfate) clearance using liquid chromatography-tandem mass spectrometric assays of serum and timed urine samples in a prospective cohort study of 298 patients with kidney disease. We estimated GFR by mean clearance of creatinine and urea from the same samples and evaluated associations of renal secretion with participant characteristics, mortality, and CKD progression to dialysis. Tubular secretion rate modestly correlated with eGFR and associated with some participant characteristics, notably fractional excretion of electrolytes. Low clearance of hippurate or p-cresol sulfate associated with greater risk of death independent of eGFR (hazard ratio, 2.3; 95% confidence interval, 1.1 to 4.7; hazard ratio, 2.5; 95% confidence interval, 1.0 to 6.1, respectively). Hazards models also suggested an association between low cinnamoylglycine clearance and risk of dialysis, but statistical analyses did not exclude the null hypothesis. Therefore, estimates of proximal tubular secretion function correlate with glomerular filtration, but substantial variability in net secretion remains. The observed associations of net secretion with mortality and progression of CKD require confirmation.

Purpose : Proteomic analysis of blood proteins in DBS is gaining attention as a possible replacement for measurements in plasma/serum collected by venipuncture. We aimed to develop and provisionally validate a nanoflow LC-PRM-MS method for clinical use. Experimental design : We used Skyline to develop a nanoflow LC-PRM-MS method to quantify glycated hemoglobin-β, apolipoprotein A-I, and apolipoprotein B in DBS. Precision, linearity, interferences, and stability and were determined and the method was used to analyze samples from 36 human volunteers. The method was compared with clinically validated measurements in paired blood collected via venipuncture. Results : The method was relatively precise for these proteins (10-11% CV) and linear across the normal concentration ranges of these proteins. Interference from high total serum protein concentration (>8 g/dL) was noted for apolipoprotein A-I and apolipoprotein B. Proteins in DBS were stable for 14 d at temperatures below 25°C and trypsinized samples were stable for 48 h at 7°C. There was moderate correlation with clinical methods (r = 0.783-0.858) and significant bias in individual samples. Conclusions and clinical relevance : Although the method had adequate precision and linearity for a biomarker, the accuracy compared with clinically validated assays raises concerns regarding the use of DBS compared with venipuncture for clinical use. This article is protected by copyright. All rights reserved

BACKGROUND Immunoassays for 1α,25-dihydroxyvitamin D [1α,25(OH)2D] lack analytical specificity. We characterized the cross-reactivity of an anti-1α,25(OH)2D antibody with purified vitamin D metabolites and used these data to map the chemical features of 1α,25(OH)2D that are important for antibody binding. Additionally, we hypothesized that when combined with isotope-dilution liquid chromatography–tandem mass spectrometry (LC-MS/MS), antibody cross-reactivity could be used to semiselectively enrich for structurally similar metabolites of vitamin D in a multiplexed assay. METHODS Sample preparation consisted of immunoaffinity enrichment with a solid-phase anti-1α,25(OH)2D antibody and derivatization. Analytes were quantified with LC-MS/MS. Supplementation and recovery studies were performed for 11 vitamin D metabolites. We developed a method for simultaneously quantifying 25(OH)D2, 25(OH)D3, 24,25(OH)2D3, 1α,25(OH)2D2, and 1α,25(OH)2D3 that included deuterated internal standards for each analyte. RESULTS The important chemical features of vitamin D metabolites for binding to the antibody were (a) native orientation of the hydroxyl group on carbon C3 in the A ring, (b) the lack of substitution at carbon C4 in the A ring, and (c) the overall polarity of the vitamin D metabolite. The multiplexed method had lower limits of quantification (20% CV) of 0.2 ng/mL, 1.0 ng/mL, 0.06 ng/mL, 3.4 pg/mL, and 2.8 pg/mL for 25(OH)D2, 25(OH)D3, 24,25(OH)2D3, 1α,25(OH)2D2, and 1α,25(OH)2D3, respectively. Method comparisons to 3 other LC-MS/MS methods yielded an r2 value >0.9, an intercept less than the lower limit of quantification, and a slope statistically indistinguishable from 1.0. CONCLUSIONS LC-MS/MS can be used to characterize antibody cross-reactivity, a conclusion supported by our multiplexed assay for 5 vitamin D metabolites with immunoenrichment in a targeted metabolomic assay.

When chronic pain patients are suspected of being non-compliant, their therapy can be withdrawn. Therefore, sensitive and specific confirmatory testing is important for identifying diversion and adherence. This work aimed to develop a novel liquid chromatography tandem mass spectrometry (LC‐MS-MS) method to detect 14 opioids and six opioid glucuronide metabolites in urine with minimal sample preparation. Analytes included were morphine, oxymorphone, hydromorphone, oxycodone, hydrocodone, codeine, fentanyl, norfentanyl, 6-monoacetylmorphine, meperidine, normeperidine, propoxyphene, methadone, buprenorphine, morphine-3-glucuronide, morphine-6-glucuronide, oxymorphone glucuronide, hydromorphone glucuronide, codeine-6-glucuronide and norbuprenorphine glucuronide. Samples were processed by centrifugation and diluted in equal volume with a deuterated internal standard containing 14 opioids and four opioid glucuronides. The separation of all compounds was complete in nine minutes. The assay was linear between 10 and 1,000 ng/mL (fentanyl 0.25‐25 ng/mL). Intra-assay imprecision (500 ng/mL, fentanyl 12.5 ng/mL) ranged from 1.0 to 8.4% coefficient of variation. Inter-assay precision ranged from 2.9 to 6.0%. Recovery was determined by spiking five patient specimens with opioid and opioid glucuronide standards at 100 ng/mL (fentanyl 2.5 ng/mL). Recoveries ranged from 82 to 107% (median 98.9%). The method correlated with our current quantitative LC‐MS-MS assay for opioids, which employs different chromatography. Internal standards were not available for every analyte to critically evaluate for ion suppression. Instead, a novel approach was designed to achieve the most rigorous quality control possible, in which the recovery of each analyte was evaluated in each negative sample.

Background The unmitigated rise in demand for the assessment of vitamin D status has taxed the ability of clinical mass spectrometry laboratories to preserve turn-around times. We aimed to improve the throughput of liquid–liquid extraction of plasma/serum for the assay of 25-hydroxy vitamin D. Methods We designed and fabricated a flexible rubber gasket that seals two 96-well plates together to quantitatively transfer the contents of one plate to another. Using the transfer gasket and a dry-ice acetone bath to freeze the aqueous infranatant, we developed a novel liquid–liquid extraction workflow in a 96-well plate format. We applied the technology to the mass spectrometric quantification of 25-hydroxy vitamin D. Results Cross-contamination between wells was ≤0.13%. The interassay imprecision over 132 days of clinical implementation was less than 10%. The method compared favorably to a standard liquid–liquid extraction in glass tubes (Deming slope = 1.018, S x|y = 0.022). The accuracy of the assay was 102–105% as assessed with the recently released control materials from NIST. Conclusions The development of a plate-sealing gasket permits the liquid–liquid extraction of clinical specimens in a moderate-throughput workflow and the reliable assay of vitamin D status. In the future, the gasket may also prove useful in other sample preparation techniques for HPLC or mass spectrometry.

The sensitive and specific detection of nicotine, its metabolites, and the tobacco alkaloid, anabasine, is useful in evaluating the success of smoking cessation treatments and detecting tobacco use, passive exposure, and nontobacco nicotine exposure in potential transplant recipients, insurance clients, and elective surgical patients. Rapid sample preparation and extended high-performance liquid chromatographic separation of tobacco alkaloids and metabolites was interfaced with tandem mass spectrometry. By using deuterated internal standards and appropriate confirmatory ion mass transitions, direct injection of centrifugally clarified urine was possible. The method had excellent precision, limit of quantitation, and linearity. The rigorous separation method revealed an interferent of nicotine that had coeluted with anabasine in more rapid chromatography and that may result in tobacco use misclassification. The method provides more specific detection of tobacco exposure and illustrates the potential of centrifugal clarification for sample preparation in the detection of multiple analytes in urine.