Tandem Mass Spectrometry Research Articles

A-042 Assessment of Known and Newly Discovered IGF-1 Variants by a High-throughput Clinical Reference Laboratory

Abstract Background The top-down approach to quantitate intact insulin-like growth factor 1 (IGF-1) by liquid chromatography coupled to high-resolution, accurate-mass, mass spectrometry (LC-HRAM-MS) is a highly specific and robust technique. The method also provides a unique opportunity to study point mutations represented as amino acid sequence variants in IGF-1. While physical consequences of most variants remain unknown, those in or close to the binding region of the IGF-1 protein (positions 30-62) may be pathogenic, while those at the C-terminus are likely benign. In addition, information about the presence of IGF-1 variants is critical for accurate estimation of growth hormone levels. In the current study, our goal was to assess the presence of new and known variants by targeted screening of a clinical population. Methods LC-HRAM-MS was used for the detection of the wild type (WT) IGF-1 and its variants in specimens submitted for IGF-1 testing. In total, 2,013,503 specimens were screened. A novel variant detection and identification (NVDI) approach was employed that uses variant group m/z, isotopic peak index (IPi), and relative retention time (rRT) to characterize IGF-1 variants. Deidentified specimens were analyzed with tandem mass spectrometry (MS/MS) and DNA sequencing to confirm variants. Results In the specimens that were analyzed, 0.44% had IGF-1 variants and 98% of these had amino acid substitutions in the C-terminal region. We identified 8 variants represented in the Exome Aggregation Consortium (ExAC) database (A67T, A70T, P66A, A67S, S34N, A38V, R36Q, and T4M), 2 previously reported variants (V44M and A67V), and 11 new variants (Y31H, S33P, R50Q, R56K, T41I, A62T, S34G, N26K, V44L, G7A, and R36W). The underlined variants listed were discovered in the current study and are first reported here. In total, 16 variants had amino acid substitutions in or close to the binding region of the IGF-1 protein, and these were found in 0.4% of patients with variants in the clinical population. Conclusions In the study population, almost all patients with IGF-1 variants had point mutations affecting the C-terminal amino acid sequence. Although these variants are likely benign, they could affect patient care if IGF-1 levels were underestimated by being based purely on WT concentrations. Pathogenic or possibly pathogenic variants occurred in a small but important fraction of the study population. The discovery of novel variants further validates this platform as a discovery engine for IGF-1 variants. We expect more variants to be discovered with more patients and broader criteria. Information about the presence of IGF-1 variants is critical for proper estimation of growth hormone levels. Patients with variants should have their physician informed to properly assess the patient’s condition. This collaboration between clinical laboratories and attending physicians may result in the characterization and better understanding of the role and effect of IGF-1 variants on growth disorders.

Nonenzymatic Oxidized Polyunsaturated Fatty Acid Products in Human Plasma and Urine Samples by LC-QTOF-MS/MS.

Oxidative stress induces autooxidation of polyunsaturated fatty acids, producing numerous isoprostanoids and isofuranoids. These oxidized products are measurable in human plasma and urine and serve as oxidative stress biomarkers for chronic diseases. This chapter details the preparation and measurement of α-linolenic acid-derived phytoprostanes and phytofurans in human samples using liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (LC-QToF-MS/MS).

B-247 A Missing Peptide Link: A Case Study Involving Detection of an Unusual IGF-1 Protein Form Using High-Resolution Mass Spectrometry

Abstract Background Previously, we described a method for monitoring insulin-like growth factor-1 (IGF-1) variants using liquid chromatography coupled with high-resolution, accurate-mass, mass spectrometry (LC-HRAM-MS). The method split s variants in 4 Variant Groups (VG) and characterizes them by the Isotopic Peak index (IPi) and relative retention time (rRT). Uncommon variants are analyzed by tandem mass-spectrometry (MS/MS) and specimens suspected to have a novel variant are deidentified and routed for DNA sequencing. A potentially novel species was detected that did not match characteristics of any previously identified IGF-1 variant. Methods LC-HRAM-MS was used for the detection of wild type (WT) IGF-1 protein and its variants in a serum specimen. Results The patient’s IGF-1 WT concentration was 210 ng/mL and z-score was -1.5, which were within normal reference ranges values for their sex and age. LC-HRAM-MS also revealed the isotopic distribution and charge for the variant, but with new characteristics (VG2:IP2, rRT= 0.01 min). The area under curve for the variant was 36% of that of the WT, which is a stark contrast to the usual 100% found in a typical heterozygous patient. MS/MS of the suspected variant showed 2 sets of b- and y-ions, which has not been seen in our laboratory. Sequencing of the fragments revealed that one set of C- and N-terminal amino acids were from the WT (C-terminus GPETL sequence and N-terminus PLKPAKSA sequence). The intact molecular weight (MW) of the species (monoisotopic m/z 1095.5250 at charge 7+) was 18 Da heavier than that of the WT (monoisotopic m/z 1092.9482 at charge 7+), so further investigation was warranted. The other set of b- and y-ions sequenced were manually identified as GL(or I)VD and PTGYG, respectively. BLAST search did not identify relevant proteins with these N- and C-termini. However, closer inspection revealed that identified sequences were found in the WT protein, albeit not at its termini. The rest of the amino acids were found that complete termini: C-terminal b-ions sequence RAPQTGIVD corresponded to positions 37-45 of the WT sequence, while N-terminal y-ions sequence KPTGYGSSSR corresponded to positions 27-36. All evidence indicates that the species is the WT protein, but with loss of a peptide bond between 2 arginines at positions 36-37 resulting in a gain of a water molecule to “cap” the newly formed termini, which is confirmed by its observed intact m/z. Conclusions We identified an extremely rare IGF-1 species formed through loss of a peptide bond between arginines at positions 36 and 37 resulting in a “missing link” in IGF-1 amino acid sequence. Despite the cleavage, the variant was a single protein molecule with intact disulfide bonds and 2 sets of C- and N-termini. The processes involved in generating this species (e.g., hydrolysis or enzymatic cleavage) and potential consequences for the patient are unknown. Because binding to receptors and binding proteins are potentially affected, and the total level of active WT IGF-1 is reduced, we are working with patient’s physician to establish whether this unusual form of IGF-1 protein may contribute to the etiology of the patient’s condition.

Global Metabolomics Using LC-MS for Clinical Applications.

Metabolomics can be used for a multitude of purposes, including monitoring of treatment effects and for increasing the knowledge of the pathophysiology of a wide range of diseases. Global (commonly referred to as "untargeted") metabolomics is hypothesis-generating and provides the opportunity to discover new biomarkers. Being versatile and having a high degree of selectivity and sensitivity, liquid chromatography-mass spectrometry (LC-MS) is the most common technique applied for metabolomics. We here present our global metabolomics LC-electrospray ionization-MS/MS method. The sample preparation procedures for plasma, serum, dried blood spots, urine, and cerebrospinal fluid are simple and nonspecific to reduce the risk of analyte loss. The method is based on reversed-phase chromatography using a diphenyl column. The high-resolution Q Exactive Orbitrap MS with data-dependent acquisition provides MS/MS spectra of a wide range of analytes. Our method covers a large part of the metabolome regarding hydrophobicity and compound class.

Potential and application of tandem mass spectrometry (MS/MS) in the analysis and identification of novel bacteriocins: a review

SummaryBacteriocins are antimicrobial peptides synthesised ribosomally by Gram‐positive or Gram‐negative bacteria to gain a competitive advantage. The majority of bacteriocins are derived from Gram‐positive bacteria, with lactic acid bacteria being the most common source. Because they are considered ‘natural’, there is currently significant development of bacteriocins for application as food preservative agents. As a preservative agent, bacteriocin activity is highly dependent on purity, down to the amino acid profile and sequence. Therefore, bacteriocin identification is important. Currently, MS is a cutting‐edge tool in bacteriocin identification. This method has high selectivity, sensitivity and resolution. To the best of our knowledge, systematic reviews focusing on the application of MS for bacteriocin identification are currently limited. In light of this, the objective of this study is to provide a comprehensive review and summary of MS technologies in bacteriocin research, with a particular focus on the discovery and characterisation of novel sources of bacteriocin. Additionally, studies related to the discovery of bacteriocins from various sources, their role as antimicrobial agents, and their synthesis are emphasised. Thus, this study presents a comprehensive analysis of the advantages, limitations, and future perspectives of the methods employed.

A-320 Determination of Guanidinoacetate Methyltransferase Enzyme Biomarkers in Newborn Dried Blood Spot by Tandem Mass Spectrometry-A Two Tier Approach

Abstract Background Guanidinoacetate methyltransferase enzyme (GAMT) deficiency is a rare, inherited metabolic disorder that affects the synthesis of creatine. Creatine is an essential metabolite for regenerating energy, particularly in the brain and muscles. Creatine is synthesized from guanidinoacetate (GUAC) by the GAMT enzyme. Mutations in the GAMT gene result in a shortage of the GAMT enzyme that causes a deficiency of creatine and accumulation of GUAC in the body, which is a neurotoxin. Following a recommendation by the Recommended Uniform Screening Panel and in line with California State statute, all California newborns will be screened for GAMT deficiency beginning July 2024. Here, we developed and validated a quantitative two-tier tandem mass spectrometry (MS/MS) assay for the detection of GAMT biomarkers (GUAC, creatine and creatinine) in newborn dried blood spot (DBS). Methods This method punches a 3.2 mm disc of controls and newborn patient DBS into a 96-well plate. 100 μL of extraction solution, containing internal standards of GAMT biomarkers, is added to each well and incubated at 40°C for 45 minutes. The extract is transferred, evaporated, and derivatized into a 3N-butanol-HCl solution at 60°C for 30 minutes. The excess HCl is evaporated to dryness. The conversion of GUAC and creatine to their butyl esters has improved their detection sensitivity by MS/MS. The butyl esters are reconstituted into the mobile phase and shaken for 10 minutes at 27°C. The final 10 μL extract was analyzed by flow injection analysis (FIA)-MS/MS in multiple reaction monitoring (MRM) positive ion mode with a total run time of 1.5 minutes per sample. The presumptive positive samples from initial analysis are prepared following the same method, and a 5 μL extract is resolved through an ultra-performance liquid chromatography (UPLC) column and analyzed by UPLC-MS/MS in MRM positive ion mode with a total run time of 3.5 minutes per sample. The validation was performed by assessing the linearity, accuracy, imprecision, carryover, limit of detection (LOD) and lower limit of quantitation (LLOQ), matrix effect, analytical measurement range (AMR), percent total allowable error (TEa), and reference range. Results This method is linear for GUAC, creatine and creatinine over a measured range of 1.17-40.00, 121.20-857.36 and 41.34-387.46 µmol/L respectively. The R2 values=1.000, 0.997, 0.9998; slope=0.999, 0.9945, 0.9982; intercept=0.0398, 4.881, 1.7027; mean recoveries=99-104%, 100-104%, 100-104%; intra-assay precision (n=20) =4.05-5.95%, 4.62-5.31%, 3.27-5.19%, inter-assay precision (n=40)=3.7-12.6%, 4.3-7.6%, 3.8-7.4%; TEa=6-21%, 7-17%, 6-17%; LOD=0.12, 0.54, 1.11 µmol/L; AMR=0.33-1300, 1.11-2700, 3.50-4500 µmol/L; median patient results observed from 2293 normal patient=1.48, 494.02, 89.97 μmol/L for GUAC, creatine, and creatinine respectively. The tier-1 and tier-2 assay screening cutoff for GUAC at the population 95th percentile is 2.53 and 4.55 μmol/L respectively. Conclusions The tier-1 and tier-2 methods has been developed and validated to determine GAMT biomarkers in newborn DBS, making it suitable for routine screening of ∼2000 newborn specimens per day by tier-1 and followed by more specific tier-2 to separate GAMT biomarkers from potential isobaric interfering compounds. This two-tier approach greatly improved the test performance and significantly reduced false positive rate.

B-162 Evaluation of two commercially available stable isotope labeled whole-protein infliximab as internal standard for a clinical infliximab LC-MS/MS assay

Abstract Background Liquid chromatography tandem mass spectrometry (LC-MS/MS) based quantitation of infliximab has been successfully introduced in the clinical laboratory for therapeutic drug monitoring in recent years. Although stable isotope labeled (SIL)-peptide or non-labeled analog protein internal standards have been utilized, SIL-whole protein infliximab is generally considered to be the ideal internal standard for the correction of variations during sample preparation and analysis. In this study, we compared two commercially available SIL infliximab proteins, Sigma SILu™MAb and Promise Proteomics, for use in a clinical infliximab assay. Methods Both labeled infliximab proteins incorporate [13C6, 15N4]-arginine and [13C6, 15N2]-lysine in their sequence and are designed for use as an internal standard for LC-MS/MS based quantitative analysis of infliximab. For the comparison of their performance, a laboratory-developed LC-MS/MS clinical method was utilized. Linearity at 6 levels in triplicate with Remicade and 3 biosimilars (-abda, -axxq, and -dyyb), intra-assay precision at 2 levels (n=10/level), and method comparison (n=15) experiments were performed using each labeled infliximab as the internal standard. For sample preparation, internal standard was mixed with the serum sample, followed by protein precipitation with ammonium sulfate, protein denaturation, and tryptic digestion. The peptides YASESMSGIPSR (YASE) from infliximab and YASESMSGIPSR [13C6, 15N4] (SIL-YASE) from labeled infliximab were monitored by LC-MS/MS (Transcend TLX and TSQ Atlis, Thermo Scientific) and utilized for the quantitation of infliximab. Results For the linearity experiment (1-50 µg/mL), while utilizing the Promise Proteomics internal standard to assay had an accuracy range of 103.4% to 114.1% for Remicade, 100.6% to 108.2% for Infliximab-axxq, 103.0% to 110.9% for Infliximab-abda, 102.7% to 108.3% for Infliximab-dyyb. For the Sigma SILu™Mab SIL infliximab, the accuracy range was 92.1% to 99.6% for Remicade, 94.5% to 107.2% for Infliximab-axxq, 99.0% to 104.2% for Infliximab-abda, and 105.1% to 112.0% for Infliximab-dyyb. The mean overall error for Remicade and biosimilars was 0.203 µg/mL (2.0%) for Sigma SILu™Mab SIL infliximab and 0.193 µg/mL (2.0%) for Promise Proteomics internal standardization. Intra-day precision using each SIL infliximab was evaluated with Remicade at low and high levels (5 and 20 µg/mL). Using Promise Proteomics, the coefficient of variation (CV) was 2.8 and 2.2%, while using Sigma SILu™Mab the precision was 6.8 and 6.5% for the low and high levels, respectively. The regression analysis for the method comparison experiment had a correlation coefficient (r) of 0.9996 with a slope of 1.050 (95% confidence interval, 1.030-1.070) and intercept of -0.318 (95% confidence interval, -0.842 to 0.205). Conclusions The mean overall error for the linearity experiment was the same using Sigma SILu™Mab or Promise Proteomics as the internal standard. The Promise Proteomics product demonstrated a lower intra-assay CV compared to the Sigma SILu™Mab. The 2 products displayed a strong correlation during the method comparison experiment. Both Sigma SILu™Mab and Promise Proteomics labeled standards showed acceptable performance as an internal standard for LC-MS/MS based quantitative analysis of infliximab. Having both products available increases the robustness of LC-MS/MS clinical assays in the event of supply issues.

A-316 A quantitative gas chromatography tandem mass spectrometry method for metabolic profiling of urine organic acids and acylglycines

Abstract Background The analysis of urine organic acids (UOA) and acylglycines (UAG) are an essential investigation for the diagnosis inborn errors of metabolism (IEMs). The vast majority of methods for these analytes use gas chromatography mass spectrometry (GC-MS) and are only qualitative. Gas chromatography tandem mass spectrometry (GC-MS/MS) offers greater sensitivity and selectivity and enables the combination of qualitative screening with quantitative analysis. We describe the development and validation results of a quantitative GC-MS/MS method. Methods Urine creatinine concentrations were measured (CobasPro, Roche Diagnostics) and normalized to 1.25 mmol/L. After internal standard addition, each urine specimen, calibration standard and QC were incubated with a hydroxylamine solution followed by a liquid-liquid extraction. Further sample concentration permitted the isolated UOA and UAG to be derivatized using N,O-bis(trimethylsilyl)trifluoroacetamine (BSTFA) with 1% chlorotrimethylsilane (TMCS) prior to injection. GC-MS/MS analysis was performed using a GC-MS-TQ8050 triple quadrupole system (Shimadzu) with a 30 m DB-5MS (Agilent J&W) capillary column (0.25 mm, ID of 0.25 µm). Split injection mode was deployed with an initial column temperature of 70°C. The mass spectrometer was operated in electron ionization (EI) and multiple reaction monitoring (MRM) modes. Results The optimal temperature gradient for high-resolution UOA and UAG separations was derived and their respective retention times determined. MS/MS detection parameters were optimized to permit the identification of precursor and production ions for all (N=36) derivatized analytes. Total analysis time was 33.8 min per injection. This method permits the measurement of 11 samples in one batch and is highly linear for all analytes (R^2 >0.99 and average slope 1.04). The limit of quantification (LOQ) was established and ranged from <0.05 to 2.1 µmol/mmol creatinine. Intra- and inter-day imprecision (CV%) was determined using matrix-matched abnormal and normal QC. Intra-day imprecision (N=10) ranged from 1.3% (2-ketoglutaric acid) to 9.7% (3-hydroxybutyric acid). Inter-day imprecision (N=30 over 15 days) ranged from 2.2% (ethylmalonic acid and methylmalonic acid) to 20.5% (3-hydroxy-3-methylglutaric acid). The method had an average accuracy of 113±14% for analytes included in the ERNDiM quantitative organic acids (urine) external quality assurance program. For analytes not included in this proficiency testing, the method showed good agreement when compared to a national reference laboratory, with average Deming correlation coefficients and regression slopes being 0.9944 and 1.1, respectively. No interferences were detected when monitoring the analyte quantifier and qualifier ion ratios in patient urine. Conclusions The devised GC-MS/MS protocol permits the unbiased identification and quantification of UOA and UAG used to investigate IEMs. The method has acceptable sensitivity, specificity, precision and accuracy for clinical routine clinical testing.

B-298 Quantitation and Abundance of Xylazine and Xylazine Metabolites in Human Urine by LC-MS/MS

Abstract Background The most common cause of drug overdose fatality in the US involves synthetic opioids such as fentanyl. Recently the Director of the White House Office of National Drug Control Policy designated fentanyl associated with xylazine as an emerging threat to the US, and a national response plan will include xylazine testing. Xylazine, a clonidine analog is not FDA-approved for use in humans but has been increasingly detected in unregulated illicit drugs supplies. Our understanding of xylazine detection, metabolism, and clinical impact on patients, however, is limited. The objectives of this study are to develop a liquid chromatography tandem mass spectrometry (LC-MS/MS) method to investigate the presence and abundance of xylazine metabolites in remnant urine samples submitted for clinical testing. Methods Remnant urine samples were identified when tested positive for xylazine during routine clinical LC-MS/MS testing. All experiments were performed using an Acquity HPLC coupled to a Waters TQS-micro triple quadrupole mass spectrometer. Urines were diluted with xylazine-D6 internal standard, hydrolyzed using β-glucuronidase and separated on a 100mm x 2.1mm, 2.6μm Kinetex C18 column (Phenomenex) with a 4 minute step gradient from 2% to 98% mobile phase B (0.1% formic acid and 2mM ammonium acetate in A: water; B: methanol) with a flow rate of 400 μl/min at 50°C. Xylazine and 4-OH-xylazine were detected in positive mode using multiple reaction monitoring, with transitions optimized from standards (Cayman Chemical and/or Sigma Aldrich), resulting in the following transitions: xylazine: 221>90, 221>164; 4-OH xylazine: 237>90, 237>137. Xylazine and 4-OH-xylazine were quantified using calibration curves prepared from 1 - 5,000 ng/mL in negative urine. Product ion scanning (PIS) was used to detect four additional xylazine metabolites, based on spectral matching to previously published full scan high resolution product ion spectra. The highest abundance fragment ions and identified retention times from PIS experiments were used to perform targeted detection for sulfone-xylazine (253>181, 253>147), oxo-xylazine (235>122, 235>114), OH-sulfone-xylazine (269>197, 269>163), and OH-oxo-xylazine (251>197, 251>148). Results Xylazine was detected at >=1 ng/mL in 38% (54/138) of fentanyl positive urines. A subset (n= 362) of urines that screened positive for opiates, amphetamines, or cocaine metabolite, were also tested but xylazine was detected in only 1 urine that did not contain fentanyl.Xylazine concentrations quantified in 61 remnant urines ranged from <1 to >5,000 ng/mL. 4-OH-xylazine concentrations ranged from <1 to 509 ng/mL. We observed five previously reported xylazine metabolites: 4-OH-xylazine, oxo-xylazine, sulfone-xylazine, OH-oxo-xylazine, OH-sulfone-xylazine. The mean concentration ratio of 4-OH-xylazine-to-xylazine was 0.11. The mean peak area ratios (metabolite-to-xylazine) for sulfone-xylazine, OH-oxo-xylazine, OH-sulfone-xylazine, and oxo-xylazine were 3.5, 3.8, 2.1, and <0.1, respectively. The proposed sulfone-xylazine and OH-oxo-xylazine metabolites were observed to have the most abundant signal of all detected metabolites, although there is currently no commercially available standard to facilitate quantification. Conclusions Xylazine and 4-OH-xylazine were quantitated in 61 patient urines. Four additional xylazine metabolites were observed and relative abundance to xylazine was estimated using peak areas. Future studies include quantitation of xylazine and xylazine metabolites in plasma and association of xylazine detection with clinical presentation and patient outcomes.

B-150 Chromatographic Separation and Quantitation of Human Insulin and Lispro Analog in Serum Using Liquid Chromatography High-Resolution Mass Spectrometry

Abstract Background Factitious hypoglycemia, a deliberate attempt to induce low blood glucose levels occurring secondary to the surreptitious administration of exogenous insulin, can be difficult to diagnose due to a variety of factors including the increased use of synthetic insulin analogs in clinical practice. Traditional insulin immunoassays demonstrate variable cross-reactivity with commonly prescribed insulin analogues and their metabolites, making it difficult to measure specific analogs. The development of liquid chromatography high resolution accurate mass (LC-HRAM)-mass spectrometry (MS) assays has largely circumvented the limitations of traditional immunoassays for detection and quantitation of insulin analogs. Analytical challenges remain, however, specifically in differentiating lispro, an isomeric human insulin analog formed by the transposition of proline and lysine near the C-terminal end of the B chain, from human insulin in patient samples due to the equivalent mass-to-charge ratios of their precursor ions. Methods Immunoaffinity purification of insulin from calibrators and quality control material prepared by spiking insulin-depleted serum with pharmaceutical insulin preparations, as well as patient samples, was performed using tips coupled with anti-insulin monoclonal antibody. An automated liquid handler was programmed to perform aspiration and dispense cycles. Eluate from the purification protocol was submitted for analysis using a TLX-2 multiplex LC system paired with a Thermo Scientific Q Exactive Plus mass spectrometer. Various HPLC column chemistries were evaluated, a column passivation procedure was established, and column heater temperature was optimized to identify a combination of parameters yielding the best chromatographic separation of human insulin and lispro. Additionally, parallel reaction monitoring (PRM) targeted tandem mass spectrometry (MS/MS) analysis was utilized in combination with the full scan data to increase specificity through the monitoring of fragment ions produced from human insulin and each insulin analog. Results We found that a 1000 Å Diphenyl, 2.7 µm, 2.1 x 100 mm analytical column in a column heater maintained at a temperature of 80°C allowed for human insulin and lispro to be differentiated as two chromatographically separated extracted ion chromatographic peaks (XICs) and quantified down to 2.5 mcIU/mL with near-baseline separation. Before use, the column required passivation with a high protein buffer (0.05% bovine serum albumin in 20% acetonitrile [ACN] + 0.2% acetic acid) followed by increasingly organic buffers (20% ACN + 0.2% acetic acid and 50% ACN + 0.2% acetic acid). MS/MS was used to confirm the presence of analogs detected. Conclusions The LC-HRAM-MS method described here, coupled with optimized column chemistry, column passivation, and proper column temperature control allows for chromatographic separation of human insulin and lispro. Incorporating a PRM scan into the mass spectrometry full scan method provides confirmation of the insulin detected in the XICs. The results obtained from preliminary studies suggest this method is appropriate for use to facilitate the clinical investigation of suspected factitious hypoglycemia with the ability to differentiate isomeric compounds, such as human insulin and lispro.

B-297 Tacrolimus Concentrations in Envarsus vs Prograf Patients: An Evaluation of the Clinical Impact of LC-MS/MS and Immunoassay Methods on Tacrolimus measurement

Abstract Background Tacrolimus is a calcineurin inhibitor used as part of an immunosuppressive regimen in kidney and liver transplant patients to prevent graft-versus-host disease (GvHD) and requires therapeutic drug monitoring due to its narrow therapeutic index. Tacrolimus levels are commonly measured using two different methodologies - immunoassay and liquid chromatography tandem mass spectrometry (LC-MS/MS). A global proficiency study to assess and compare the measurement of trough levels by these two methodologies noted a positive bias with the ARCHITECT immunoassay which is likely due to the presence of tacrolimus metabolites. The aim of our study is to assess the impact of two different formulations of tacrolimus on the accuracy of its analytical measurement. While differences between immunoassay and LC-MS/MS results have been evaluated for immediate-release tacrolimus (Prograf), it has yet to be characterized for extended-release formulations such as Envarsus. The discrepancy between immunoassay and LC-MS/MS has clinical implications, as dosage modifications are based on measured concentrations, analytical variation may cause intervals of subtherapeutic or supratherapeutic exposure, increasing the risk of graft rejection or toxicity. Methods Development of an LC-MS/MS method for the simultaneous quantification of tacrolimus and its major metabolite, desmethyl tacrolimus, was performed in whole blood. Traceable calibrators and quality control material for tacrolimus and specimens supplemented with desmethyl tacrolimus were used to determine accuracy and precision. Tacrolimus concentrations were measured by LC-MS/MS and ARCHITECT immunoassay methods in excess whole blood specimens from patients treated with either Prograf or Envarsus. Measurement biases between LC-MS/MS and immunoassay methodologies were determined for both formulations of tacrolimus. Results External calibration curves for both tacrolimus and desmethyl tacrolimus were linear (R2>0.995), and the analytical measurement range (AMR) for tacrolimus spanned from 1.1 - 31.6 ng/ml. Calibrator and Quality control (QC) biases were within 15% of their target values throughout the AMR and within-run imprecision was less than 10% (n=25) for all calibrators and QCs. Between-run imprecision for Low, Mid, and High QC levels over a period of 2 weeks (n=5 days) was less than 10%. Comparative bias of tacrolimus concentrations between immunoassay and LC-MS/MS was significantly lower (P value=0.0074) for Envarsus (n=20 specimens) relative to Prograf (n=32 specimens). Conclusions The differential bias between immunoassay and LC-MS/MS in the measurement of tacrolimus in patients dosed with immediate-release versus extended-release formulations suggests that their distinct pharmacokinetic profile may impact the accuracy of the measurement. Therefore, applying observed measurement biases from different assays derived from Prograf patients to Envarsus patients and vice versa may result in erroneous estimation of tacrolimus concentrations.

B-249 Assessing Targeted Mass Spectrometry Assay Performance for Putative Biomarker Proteins on a New Hybrid Nominal Mass Instrument

Abstract Background The development of targeted mass spectrometry-based proteomics assays that monitor biomedically relevant proteins is an important step in bridging discovery experiments to highly multi-analyte clinical studies. Targeted assays are currently unable to scale to thousands of peptide targets from hundreds of proteins, requiring extensive refinement down assays for the minimum useful analytes. Large parallel reaction monitoring (PRM) assays are now possible with a new hybrid nominal-mass instrument. The scale of assay is achievable with this instrument by using real-time alignment, while being sensitive and fast enough to handle many concurrent targets. To assess the performance of this new instrument we constructed a large scale PRM assay for proposed neurodegenerative disease marker proteins, and an assay for proteins associated with amyloid tissue typing. Methods To evaluate the performance of this instrument on proteins of interest in cerebrospinal fluid, we performed an analysis on a pool from individuals diagnosed as Alzheimer’s, Parkinson’s, or cognitively normal. A calibration curve of cerebrospinal fluid diluted into chicken serum at 14 dilutions and individual human cerebrospinal fluid were digested with a protein aggregation and capture based digestion protocol. CSF peptides were separated by 60 minute gradient on C18 IonOpticks column, and amyloid tissue peptides separated by 30 minute gradient on C18 PepMap column with a Vanquish Neo UHPLC and analyzed by data-independent acquisition (DIA) and PRM with a new hybrid nominal mass instrument designed for targeted tandem mass spectrometry (tMS2). Scheduled methods were generated using a Skyline external tool, transitions were refined to minimize interference and maximize the LLOQ. DIA data was searched, and data extracted and analyzed in Skyline. PRM data was extracted and quantified with Skyline; CSF calibration curve LLOQ is calculated by bilinear fit. Results For the large scale cerebrospinal fluid (CSF) assay the selection of protein targets was based on literature from neurodegenerative disease cohort studies. We target 1065 peptides from 104 proteins, including proteins currently measured by ELISA in clinical research, e.g.: APP, TAU, and APOE. Sensitivity and reproducibility of the assay is assessed using figures of merit from replicate matched-matrix calibration curves. Over 80% of proteins have at least one peptide with a LLOQ below 30% dilution in the matched matrix, and of all targeted peptides over 55% had a LLOQ below 30% dilution. To demonstrate the application of the real-time alignment on the new instrument we constructed an assay for samples where not every analyte is shared. An assay for amyloidosis associated proteins was constructed for a total of 180 peptides mapping to 52 proteins. This includes 11 proteins most commonly associated with amyloid deposition; APCS, APOA4, APOE, CLU, LECT2, IGKC, IGLC2/4/7, SAA1, TTR, and VTN. From the preliminary set of 12 previously typed samples we observe peptide abundances consistent with the assigned type. Conclusions This novel instrument provides a new approach for building large targeted mass spectrometry assays. We demonstrate the ability of the real-time alignment to adjust scheduled retention time windows, ensuring that chromatographic shifts do not lead to a loss of peptide detection and quantification.

B-255 Identification of the protein expression of Androgen Receptor variants using Targeted proteomics in clinical Castration Resistant Prostate Cancer models

Abstract Background Castration Resistant Prostate Cancer (CRPC) is a treatment resistant form of prostate cancer (PCa). Currently, there is not a way to identify which patients will develop this resistance before full blown CRPC develops. Therefore, all PCa treatment approaches are similar yet this results in tumor regression in some cases and progression in others. Targeting the androgen receptor (AR) is still the main focus of current therapies even in CRPC. Emergence of AR splice variants (AR-Vs) after initial treatment is thought to be one of the primary mechanisms of resistance. AR-Vs lack the ligand binding domain rendering Androgen Deprivation Therapy (ADT) ineffective in tumors expressing these variants. Recent work has identified the DNA and RNA species of ARVs in CRPC but investigation into whether the protein is translated are unknown. One exception is the approval of an antibody test that detects a specific AR-v, AR-v7, from the blood of PC patients and predicts ADT response. However, there are instances where a patient may not express ARv7 and are still resistant to ADT. This may suggest that other AR-Vs, not currently detected at the protein level, are important predictors for ADT response. Methods The primary goal of this work is to expand the protein identification of known and unknown AR-Vs that may predict response to ADT using targeted liquid chromatography tandem mass spectrometry (LC-MS/MS). We developed the AR-V targeted LC-MS/MS assay by first designing AR splice variants from unique splice regions via RNA-guided sequences translated to amino acid sequences. The AR-variants full amino acid sequences were then in-silico trypsin digested using Expasy software tool that generated tryptic peptides. Blast analysis of the AR-V peptides was performed where no overlap with other human proteins was observed. These AR-V peptide sequences were commercially synthesized along with heavy isotopically analogs to be used as internal standards in the LC-MS/MS method. The method examined 9 peptides and included 2 to 3 MRM transitions per peptide and calibration curves were constructed using unlabeled peptides. Specimens examined consisted of a panel of PCa cell lines (n= 6), CRPC patient derived xenografts tumors (PDX) (n=4) and a negative control cell line (no androgen receptor expression), Cos-1, which served with a dual function and was used as matrix for peptide MRM optimization experiments. Linearity and sensitivity for each peptide included in the method was examined using calibration curves. Results The targeted MRM approached was successfully achieved for the quantification of 9 peptides from 8 proteins in 6 PCa cell lines and 4 PDX tumor specimens. Out of the 8 proteins evaluated, we observed 3 known targets (AR-exon1, AR-V7 and AR-V12) and 4 novel AR-Vs in 22Rv1 PCa cell lines. Additionally, CRPC PDX were assessed where 3 novel AR-Vs were detected. Conclusions These preliminary results using a LC-MS/MS platform show promising identification and quantification of novel AR-Vs that have not been measured before at the protein level. Subsequent analysis of larger cohorts will further elucidate AR-Vs role in PCa treatment resistance mechanisms and possibly assist in future treatment approaches.

B-173 Cystine Analysis in Urine Sample Using the Laser Diode Thermal Desorption technique “LDTD”/Mass Spectrometry (Luxon)

Abstract Background Cystine is a polar amino acid found in the urine of healthy individuals; however, a genetic defect in renal transport can lead to cystine crystals and stones. A usual laboratory approach for diagnosing and monitoring cystinuria is to measure cystine concentration in 24-hour urine. The disease is rare, affecting around 1 in every 10,000 live births. The objective of this study was to develop a rapid assay using Tandem Mass Spectrometry methodology with an internal standard of Cystine derivatized in a urine sample, Luxon Ion Source®, Laser Diode Thermal Desorption (LDTD) technology. Methods The Luxon is a sample introduction and ionization source with a coupled laser diode, providing thermal uniformity, precision, accuracy and speed. Thermally desorbed neutral molecules are analyzed by mass spectrometry (MS). Initially, the sample was derivatized to make the cystine thermally vaporize, followed by liquid-liquid extraction assisted by salting out (SALLE). The isotope-labeled internal standard (Cystine-d4), homemade calibration curve and water quality controls were used for quantification. The analysis was performed using a Sciex 6500+ Triple Quad in APCI (Atmospheric Pressure Chemical Ionization) positive mode. The ratio of peak area to internal standard (ISTD) was used to normalize the signal. A linear regression model with 1/X2 weighting was used to obtain the calibration curve equation and determine the correlation coefficient (r2). Parallelism was assessed by comparing the (r2) of calibration curves prepared in water with six curves prepared with biological matrices. Intra- and interassay precision and accuracy were determined by measuring control samples ranging from low to high concentration in five replicates over 3 consecutive days. For urine analysis, aqueous calibration curves in the concentration range of 1 to 100 mg/L (n=3) showed excellent linearity. The following parameters were evaluated for quantitative analysis: linearity, precision, accuracy, carryover and parallelism.​ Results The calibration curve resulted in a linear response across the entire concentration range, the correlation coefficient (r) was 0.99. For each quality control concentration, CVs were <10%, meeting desirable imprecision and precision specifications. The intraassay CVs were 1.0-2.56% and the interassay CVs were 2.15-4.58% for precision, which reflects the agreement between repeated measurements. Precision results showed similar results, intra-assay CVs were 2.23-3.66% and inter-assay CVs were 1.0-2.77% Conclusions The new LDTD-MS/MS method for the determination of urinary Cystine was accepted after verification and validation of quality control parameters. The technique proved to be effective for reliably and quickly quantifying cystine in urinary samples from human patients, being a valuable tool in the diagnosis and prevention of cystine kidney stones. The assay developed, using the Luxon Ion Source®, based on LDTD (Laser Diode Thermal Desorption) technology, proved to be ultrafast for measuring derivatized cystine in urine

B-302 Therapeutic Drug Monitoring (TDM) of Hydroxychloroquine in Whole Blood: Analysis of Over 10,000 Patient Results

Abstract Background Hydroxychloroquine (HCQ), a mainstay of systemic lupus erythematosus (SLE) therapy, improves survival and reduces flares. Therapeutic drug monitoring (TDM) may be useful in 1) identifying and improving adherence issues, 2) titrating daily dose to achieve therapeutic benefits while 3) minimizing retinopathy due to longterm cumulative exposure. Whole blood (WB) is the correct specimen for HCQ TDM because WB HCQ levels are higher than serum or plasma levels, more stable and reflective of the last month of HCQ ingestion, and are better correlated to efficacy and retinopathy risk. Here, we report WB HCQ concentrations in 10,463 clinical patient samples and their distribution. Methods HCQ concentrations in whole blood are measured by liquid chromatography tandem mass spectrometry (LC-MS/MS) and reported in nanograms per milliliters whole blood. Recommended sample collection is ∼ 3 - 6 months into regular stable dosing to reach pharmacokinetic steady state and correlate with clinical efficacy. Results Here, we report the analysis of 10,463 patient samples for whole blood hydroxychloroquine (WB HCQ) concentrations. More than one-third (3752, 35.9%) were within the target range of > 1000 and < 2000 ng/mL. About one-quarter (2568, 24.5%) were within a tighter therapeutic target window, 1000 to 1500 ng/mL. The vast majority (9852, 94.2%,) of all WB HCQ were < 2000 ng/mL with a small percentage >2000 ng/mL (611, 5.8% between 2000 - 9502 ng/mL). About one-third (31.3%) were higher than 1177 ng/mL which corresponds to higher retinopathy risk. Adherence issues were evident in 10.5% (1102) with undetectable (483, 4.6%) and very low (<200 ng/mL) drug (619, 5.9%) (yellow). Depending upon the target threshold, levels < 500 ng/mL or <1000 ng/mL may be considered subtherapeutic as seen in 2498 (23.9%) and 4263 (40.7%) samples, respectively. Conclusions Due to wide pharmacokinetic variability and adherence issues, a given prescribed dose of HCQ does not correspond to WB HCQ, e.g. following 4, 5 or 6 mg/kg/day, levels are known to vary widely from subtherapeutic <500 to >2000 ng/mL. Here, our clinical database reiterates this wide range in WB HCQ levels from undetectable (<25) to 9502 ng/mL in 10,463 patient samples. One-quarter or one-third were within the therapeutic target range of 1000-1500 or 1000-200 ng/mL where >1000 has been associated with better disease control and survival. At the same time, levels less than 1177 avoid higher retinopathy risk. A small percentage of samples, 5.8%, were higher than 2000 ng/mL, suggesting the usefulness in TDM to fine-tune daily dose. In 10%, there was evidence of poor adherence, with very low or no detectable drug, and one quarter to 40% were subtherapeutic (<500 or <1000). The potential benefits of HCQ are many and include reduced flares and improved lupus nephritis, thrombosis risk, and long-term survival. At the same time, prescribed dose does not predict therapeutic levels. TDM-based counseling and dose adjustment may be instrumental in optimizing HCQ benefits while minimizing retinopathy risk. In conclusion, our analysis demonstrates the growing use of whole blood HCQ TDM to facilitate dose titration to safe and most effective target levels.

A-075 Comparability of Free Testosterone Measurements- Results of the Interlaboratory Study

Abstract Background Assessment of free testosterone (FT) has been recommended as a biomarker for diagnosis and management of hypogonadism and female hyperandrogenism. Reliable laboratory measurements are essential for correctly diagnosing and treating these diseases. FT is currently measured using direct commercial immunoassays, assays employing equilibrium dialysis (ED) or other separation techniques, or it is estimated by calculating free testosterone (cFT). There are concerns about the accuracy and reliability of current methods that may impact patient care. To obtain more information about variability of FT measurements used in patient care, CDC’s Clinical Standardization Programs (CDC CSP) conducted an interlaboratory comparison study. Methods CDC CSP developed an accurate, automated method using ED coupled with isotope dilution ultra-high-performance liquid chromatography tandem mass spectrometry (ED-UHPLC-MS/MS). The ED step follows an internationally recognized procedure. The method is calibrated with primary reference material (National Measurement Institute-M914) and was used to assign target values to sera used in the study. The FT concentrations ranged from 3 to 339 pg/mL. In the interlaboratory comparison study single-donor serum samples were provided to participants operating FT methods used in patient care. The participating methods included commercially available platforms utilizing Chemiluminescence Immunoassay or radioimmunoassay techniques, as well as laboratory-developed assays using ED-LC-MS/MS. Results Preliminary findings of the CSP interlaboratory comparison study found large variability among participating assays. The agreement among IA and among ED-LC-MS/MS assays was close. However, FT concentrations obtained by IAs were in average 6 times lower compared to the ED-LC-MS/MS methods. Results estimated using the Vermeulen equation overestimated FT in average by 15% compared to the ED-LC-MS/MS methods and by over 6 times compared to IAs. Conclusions Large variability is observed between FT laboratory developed tests (LDTs) and IAs. As both LDTs and IAs are used in patient care, standardization of FT measurements to assure better patient care is needed. CDC CSP is addressing the need by developing highly accurate and precise FT method and conducting an interlaboratory comparison studies that can aid with improvement of the agreement among assays and identifying potential factors causing differences among assays.

B-156 Extraction strategy for therapeutic drug monitoring of sirolimus and tacrolimus using volumetric absorptive microsampling

Abstract Background Following organ transplantation, patients are placed on immunosuppressant drugs (ISDs) to minimize the risk of rejection. Sirolimus and tacrolimus are two of the most widely used ISDs, however, due to their narrow therapeutic indices, patients require frequent therapeutic drug monitoring (TDM). Recurrent venipuncture can become uncomfortable and disruptive, especially in the pediatric population. Therefore, we describe an extraction method using dried whole blood from volumetric absorptive microsampling (VAMS) devices for the detection of sirolimus and tacrolimus by turbulent flow liquid chromatography tandem mass spectrometry (TFC-MS/MS). The use of VAMS in the clinical laboratory will provide ISD patients with a convenient alternative to traditional blood draws. Methods MassTrak immunosuppressant calibrators based on a whole blood matrix (Waters) were applied to 30 μL VAMS Mitra devices (Neoteryx) and dried overnight. To extract the whole blood off of VAMS tip, we tested various methanol:water solvent ratios, of which 30 μL was added to the device. Tips were extracted using ultrasonication (30 min), vortexing (5 min), and centrifugation. Once extracted from the tip, 25 mM zinc sulfate was added to whole blood extract at a 1:1 (v/v) ratio. An acetonitrile and methanol extraction reagent was then added to the whole blood/zinc sulfate solution at a 2:1 (v/v) ratio. After brief vortexing, samples were centrifuged at 16,060 × g (10 min) and tested using an established TFC-MS/MS method which deploys a TLX-2 system for sample preparation coupled to a Q-Trap tandem mass spectrometer (AB Sciex 5500). Results A 80:20 (v/v) water:methanol extraction solvent yielded the greatest extraction efficacy for both sirolimus and tacrolimus from the VAMS tips compared to other tested ratios. We established a 5-point calibration curve to correlate the measured values of ISDs with known MassTrak calibration standard concentrations. The resulting average calibration curve from 3 independent experiments was highly linear for both sirolimus (R2= 0.992; slope= 1.1; intercept= 0.94) and tacrolimus (R2= 0.996; slope= 1.1; intercept = 0.8819). Our VAMS whole blood extraction strategy was correlated to the established neat whole blood protocol, which utilized all of the sample preparation steps outlined above except the initial extraction from the VAMS tip. The average extraction efficacy was 89.1% for sirolimus with an average coefficient of variation (CV%) of 12.1% across 3 independent experiments. For tacrolimus, we demonstrated an average extraction efficacy of 100.1% with an average CV% of 8.4%, again across 3 independent experiments. Conclusions We demonstrate the feasibility of using a VAMS approach to TDM of sirolimus and tacrolimus. Our results demonstrate favorable recoveries for both of the tested drugs, evidenced by high recoveries of the external calibration standards. Additionally, our method exhibited high extraction efficacy and low variability. Further validation studies, including the utilization of clinical samples is necessary to confirm our preliminary findings.

B-260 Prevalence and Relative Distributions of Major Phosphatidylethanol (PEth) Homologs in a Large-Scale Clinical Study

Abstract Background Ethanol abuse remains a significant public health challenge in the United States. Detection of ethanol exposure is crucial for preventive, diagnostic, and therapeutic endeavors. Phosphatidylethanol (PEth) is a specific and direct ethanol biomarker. Compared to other metabolites, such as ethyl glucuronide and ethyl sulfate in urine (average 2-3 days), PEth in blood has a longer window of detection (average 1-2 weeks). Among the known 48 PEth homologs, PEth 16:0/18:1 (POPEth) and PEth 16:0/18:2 (PLPEth) are the most abundant species accounting for 37% to 46% and 26% to 28%, respectively. PLPEth is synthesized at a greater rate than POPEth, but POPEth has a longer half-life than PLPEth. The purpose of this study was to understand the prevalence and levels of POPEth and PLPEth in a large number of clinical specimens. Methods Briefly, lavender-top whole blood specimens were diluted with isotopically labeled internal standard (IS) in an organic buffer to precipitate proteins on an automated Hamilton platform. The mixture was centrifuged, and a supernatant portion isolated for injection onto a Sciex QTRAP 5500 liquid chromatography tandem mass spectrometry (LC-MS/MS) system using electrospray ionization (ESI) in negative mode. The assay was fully validated under CLIA regulations for lab developed tests: the analytical measurement range (AMR) was 10-400 ng/mL; the administrative reporting threshold for PEth positivity was 20 ng/mL based on clinical literature and a consensus among other United States laboratory methods. Results Precision of 4 QC levels was evaluated with between-run CV% (n=25) ranging from 1.6-6.3%, and accuracy across the AMR was achieved with 87-110%. No significant matrix effects were observed; variation in different whole blood specimens was corrected with IS normalization. No significant interferences were observed from 137 commonly seen prescription and nonprescription drugs and 6 other PEth homologs. Of the clinical specimens, most (20,833, 71.3%) were negative (<20 ng/mL) for both analytes. Meanwhile, 8,222 specimens (28.3%) were positive with either POPEth, or PLPEth or both ≥ 20ng/mL. There were 150 specimens excluded from the statistics due to interference challenges. Interestingly, 757 specimens (2.6%) were PLPEth negative and POPEth positive (mean value 40.1 ng/mL). Conversely, 62 specimens (0.2%) were POPEth negative and PLPEth positive (mean value 24.6 ng/mL). Among the specimens with both POPEth and PLPEth levels within the AMR, the average POPEth/PLPEth ratio was 1.4 supporting the overall prevalence of higher POPEth concentrations than PLPEth. However, the POPEth/PLPEth ratio varied widely, from 0.3 to 8.8, reflecting patient differences in PEth metabolism patterns. Conclusions To help verify probable ethanol abstinence, we have developed, validated, and deployed a high-throughput clinical assay for the major PEth homologs, POPEth and PLPEth. In a retrospective analysis of 29,205 clinical specimens: 71.3% of results were consistent with abstinence; 28.3% of the results were positive suggesting moderate alcohol consumption within the last 1-2 weeks. With POPEth most prevalent, yet variation observed, there were 62 specimens with PLPEth positive and negative for POPEth. This clinical PEth study including both POPEth and PLPEth provides greater sensitivity to identify ethanol exposure than only testing a single homolog.

A-067 Simultaneous Measurement of Aldosterone and Plasma Renin Activity in Human Serum Using Liquid Chromatography Coupled to Tandem Mass Spectrometry for Clinical Research

Abstract Background The combination of primary aldosteronism (PA) and alterations in plasma renin activity (PRA) is a leading cause of secondary hypertension. An accurate measurement of Renin Angiotensin-Aldosterone System (RAAS) pathway improves the recognition of symptoms related to stroke, coronary disease, heart failure and metabolic syndrome. Methods Previously, surrogate matrices have been used when demonstrating the analytical performance of LC-MS/MS clinical research methods for the measurement of aldosterone and angiotensin I. Here, we have developed an analytically sensitive and reliable analytical strategy for simultaneous measurement of Aldosterone and Angiotensin I in human matrices. In-house calibrators and quality control samples were prepared, containing varying ranges of both aldosterone (20-2000pg/mL; 55-5548pmol/L) and angiotensin 1 (0.6-240ng/mL; 0.46-185nmol/L). The samples were extracted using the Oasis™ MAX 96-well µElution plate and then separated through the ACQUITY™ UPLC™ I-Class FL System using a XBridge™ Premier Peptide BEH™ C18 Column, over a run time of 3 minutes. Samples were then injected to Xevo™ TQ Absolute Mass Spectrometer, using both positive and negative acquisition modes. The downstream LC-MS/MS data analysis was performed using MassLynx™ MS Software. The analytical performance was demonstrated by extracting and quantifying two replicates of samples on two occasions per day over five separate days. Additionally, the stability of aldosterone and angiotensin I over the period of incubation (3-5hr) was evaluated, which is necessary when investigating the plasma renin activity in human Serum/Plasma samples. Results High reproducibility and repeatability were both determined, indicating total precision and repeatability (<15%CV) for both analytes and no significant changes were found in Aldosterone and Angiotensin I concentrations over 3-5hr of incubation. No significant system carryover (<10% of the lowest calibrator) from high concentration samples into subsequent blank injections was observed. In addition, calibration lines in human serum/plasma were linear, with coefficient of determinations (r2) >0.992 for all analyses at different incubation times. The analytical sensitivity of the clinical research method allows for precise quantification at 55 pmol/L for aldosterone and 0.46 nmol/L/hr for angiotensin I, with both analytes providing S/N (PtP) ≥10:1 and %RSD of <15% at these concentrations. Conclusions An analytically sensitive and selective clinical research method has been developed for the analysis of aldosterone and angiotensin I, using the Xevo TQ Absolute Mass Spectrometer. The Xevo TQ Absolute Mass Spectrometer enables the simultaneous analysis of physiologically low levels of aldosterone and plasma renin activity in human samples, (20pg/mL; 55pmol/L), (0.6ng/mL; 0.46nmol/h/L). A single test that improves instrument utilization, reduces cost, and allows the user to expand their existing test menu on the same platform has been established.

B-182 Development and validation of an LC-MS/MS assay for simultaneous measurement of serum androstenedione and 17-hydroxyprogesterone in serum

Abstract Background Androstenedione and 17-hydroxyprogesterone are measured via immunoassays or high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) in clinical laboratories. Immunoassays have limited sensitivity and specificity for certain steroid hormones testing. We aimed to develop and validate a sensitive, accurate, and specific in-house LC-MS/MS assay to simultaneously measure both 17-hydroxyprogesterone and androstenedione in serum. Methods 13C-labeled androstenedione and 17-hydroxyprogesterone internal standards were added to calibrator, control, and patient samples. A hexane/ethyl acetate solution was used in a liquid-liquid extraction procedure. The organic layer was then removed and dried followed by reconstituting the extracts in 2.5 mM ammonium formate in 50% methanol. The HPLC mobile phase gradient began with 47%, 2.5 mM ammonium formate in water and 53%, 2.5 mM ammonium formate in methanol. This progressed to 56% of the methanol phase over three minutes. The mixtures were separated though an Agilent 1260 Infinity HPLC system with a Poroshell 120 EC-C18 analytical column (2.1 × 50 mm × 2.7 µm). The separated HPLC eluents then entered an Agilent 6460C QQQ mass spectrometer in positive ion mode through an electrospray ionization source where protonated androstenedione (m/z = 287.2) with the protonated androstenedione-[2, 3, 4-13C3] internal standard (m/z = 290.2) and protonated 17-hydroxyprogesterone (m/z = 331.2) with the protonated 17-hydroxyprogesterone-[2, 3, 4-13C3] (m/z = 334.2) were mass-selected for collision-induced dissociation. For androstenedione, respective quantifier and qualifier m/z values of 287.2 > 97.1 and 287.2 > 109.1 were used while respective quantifier and qualifier m/z values of 331.2 > 97.1 and 331.2 > 109.1 were used for 17-hydroxyprogesterone. Validation studies were performed including imprecision, lower limit of quantitation (LLOQ), analytical measurement range (AMR), interference, specificity, carryover, extraction recovery, matrix effect, and comparison with a reference laboratory LC-MS/MS method. The total allowable error limits used in this study were 23.5% for androstenedione and 30.2% for 17-hydroxyprogesterone. Results Total imprecision results across three concentrations yielded a maximum CV of 4.6 % for androstenedione and 6.5% for 17-hydroxyprogesteron. The AMRs of androstenedione and 17-hydroxyprogesterone were 0.2 to 13.4 and 0.1 to 22.0 ng/mL without significant carryovers. No significant interferences were observed from 11-deoxycorticosterone (700 ng/dL), testosterone (2500 ng/dL), DHEA (1000 ng/mL), bilirubin (30.1 mg/dL), hemoglobin (1032 mg/dL), or triglycerides (1858 mg/dL). The extraction recoveries of androstenedione and 17-hydroxyprogesterone were 98% and 81%, respectively. No significant matrix effects were identified. In comparison to the reference lab LC-MS/MS method, the in-house LC-MS/MS assay demonstrated a 0.523% bias for androstenedione and a -3.77% bias for 17-hydroxyprogesterone with both exhibiting excellent correlations with the reference lab measurements. More specifically, our results (as a function of values obtained from the reference lab) followed the relationship of y = 1.039x - 0.092 (R = 0.9951) for androstenedione and y = 0.957x + 0.01 (R = 0.9974) for 17-hydroxyprogesterone. Conclusions An LC-MS/MS assay is developed for simultaneous detection of serum androstenedione and 17-hydroxyprogesterone. The assay demonstrates acceptable imprecision, AMR, and accuracy. No significant carryover and interference from other structurally similar steroid hormones and other common interferents are identified.