Podium Presentations for Troubleshooting

Is the Fentanyl Result Real? Tackling Interfering Substances Amid the Opioid Epidemic Hsuan-Chieh (Joyce) Liao (Presenter) University of Washington

To be presented in Track 4 (Colton) on Wednesday at 13:30

INTRODUCTION: The surge in illicit fentanyl use has significantly escalated opioid-related overdose fatalities. In response to the ongoing opioid epidemic, clinical laboratories are increasingly incorporating fentanyl-specific testing into their urine drug screening panels, utilizing both immunoassays and LC-MS/MS assays.

OBJECTIVES: This practical training aims to outline the strengths and limitations of LC-MS/MS in detecting fentanyl, offering insights into best practices through case studies and discussions.

METHODS: While immunoassays are cost-effective and rapid, making them ideal for initial screenings, LC-MS/MS is predominantly employed as a confirmatory test due to its superior sensitivity and specificity. However, both immunoassays and LC-MS/MS assays can yield "false positive results" due to interfering substances. High-resolution mass spectrometry may be helpful in identifying these interferences.

RESULTS: Structurally similar compounds, such as fluoroquinolone antibiotics (e.g., ciprofloxacin, ofloxacin, and their metabolites), can trigger cross-reactivity in fentanyl immunoassays. While "false positive results" in LC-MS/MS are uncommon, they can occur due to interfering substances from system reagents, instrument fluidics, or the sample itself. Detecting and resolving these issues requires systematic and logical troubleshooting approaches.

CONCLUSION: The use of LC-MS/MS as confirmatory testing, along with high-resolution mass spectrometry, can effectively identify interfering substances in immunoassays. To ensure accurate drug testing LC-MS/MS results, it is essential to continuously monitor quality metrics and employ strategic troubleshooting to effectively manage and mitigate potential interferences.

A C18 is a C18 is a C18…? Effects Of Different Types Of C18 Columns On The Separation Of Bile Acids From Interferences In Matrix Samples Kat Iacob (Presenter) ARUP Institute for Clinical and Experimental Pathology

To be presented in Track 3 (Steinbeck 3) on Thursday at 11:10

INTRODUCTION: Bile acids (BAs) have diverse biological functions in lipid digestion and cell signaling. Their composition and concentration changes in various acute and chronic health conditions, and in response to therapies. Therefore, accurate quantitation of individual BAs is of clinical significance. Nowadays, it is usually achieved by LC-MS/MS analysis. Clinically relevant bile acids are characterized by several isomeric compounds which need to be separated. There are also BAs that do not fragment in the mass spectrometer (or only poorly so). Due to their chemistry, BA analysis in patient samples can be subject to matrix interferences which cannot easily be removed or avoided, and which can greatly affect quantitation of the analytes.

OBJECTIVES: The main objective of this study was to develop an LC method for quantitative BA analysis that minimizes matrix interferences on 13 analytes and their respective IS in patient samples.

METHODS: Six different LC columns with C18 chemistry from four different manufacturers were used with four different mobile phase systems to analyze patient samples (six total combinations). To test contribution of sample preparation to matrix interference removal, simple protein crash methods (acetonitrile or methanol) were tested and compared to several SPE products. Chromatographic separation of analytes and possible interferences was compared between LC systems using non-matrix and matrix samples.

RESULTS: Regardless of column, neutral/acidic pH mobile phases were superior to basic pH mobile phase in separating bile acids, allowing full baseline separation of the 13 analytes into 13 peaks. Removal of matrix interferences was not improved by engaging SPE sample preparation methods compared to an acetonitrile or methanol protein crash. All tested methods gave good linearity over the desired AMR (r2 ≥ 0.99) with neat standards and performed well with spiked controls. But only two combinations of C18 column and mobile phase system were successful at consistently separating interferences from analyte and IS peaks in matrix samples.

CONCLUSION: Due to the chemistry of bile acids, quantitative LC-MS/MS analysis of BAs requires a specific combination of column and mobile phases to successfully separate analyte and IS peaks from interference peaks.

Poster Presentations for Troubleshooting

Positive Bias in Fractionated Vitamin D2-D3 Method as Determined via LC-MS/MS Elizabeth Mast (Presenter) IU Health

Poster #1a View Map

This poster will be presented and discussed on Wednesday at 17:45 for 15 minutes in Montreal 3 (Track 2).

1. Problem
Method comparison of 25-hydroxyvitamin D3 showed significant number of samples with >20% positive bias while compared to reference method during validation of new instrumentation in the laboratory while 25-hydroxyvitamin D2 was within acceptable limits.

2. Method Information
200 uL serum sample prepared by acetonitrile protein precipitation.
TSQ Altis with Transcend TLX-LC; APCI
Turbo column: C18 XPL 0.5x50 mm
Turbo Mobile Phases: A- 0.1% Formic acid/ Water, B- 0.1% Formic acid/ Methanol; C- 40/40/20 ACN/IPA/Acetone
Analytical column: C18 Accucore 2.6 um 50 x 3mm
Analytical Mobile Phases: A- 0.1% Formic acid/Water, B- 0.1% Formic acid/Methanol

3. Troubleshooting Steps
Analysis of NIST standards, particularly the NIST standard with >20% epimer, showed the method was unable to separate 25-hydroxyvitamin D3 from 3-epi-25-hydroxyvitamin D3, thus showing >20% positive bias in the patients samples. We developed a new LC gradient using fluorophenyl 2.7um 100x3 mm column that was able to obtain baseline resolution of 25-hydroxyvitamin D3 from 3-epi-25-Hydroxyvitamin D3. Further verified accuracy using CAP PT samples.

4. Outcome
The new flurophenyl method was able to obtain baseline resolution of 25-hydroxyvitamin D3 from 3-epi-25-hydroxyvitamin D3 while maintaining the extra online extraction provided by the turbo column on the TLX Transcend HPLC. While this method is longer, performance of the new method resolved the positive bias in 25-hydroxyvitamin D3 as compared to the reference method. Additionally, the number of samples with detectable 3-epi-25-Hydroxyvitamin D3 corresponded with the number of samples with >20% bias seen during previous validation studies. While 3-epi-25-Hydroxyvitamin D3 was not quantitated, Tracefinder, data analysis software, allows for an estimation of the amount present, which was in agreement with NIST standards with known 3-epi-25-hydroxyvitamin D3 values. Separation of 25-hydroxyvitamin D2 from 3-epi-25-hydroxyvitamin D2 was also achieved. No significant amount of 3-epi-25-hydroxyvitamin D2 was seen in patient samples.

Ordeals of Developing a Method to Measure Low-Level Concentrations of Serum Testosterone and my Troubleshooting Journey Leslie Farris (Presenter) Cleveland Clinic Foundation

Poster #1b View Map

This poster will be presented and discussed on Wednesday at 18:00 for 15 minutes in Montreal 3 (Track 2).

1. Problem

We needed to improve sensitivity to measure low concentrations (down to ~1 ng/dL) of total testosterone in serum by LC-MS/MS. Several issues affecting sensitivity and accuracy (to a lesser extent) were encountered throughout method development, as listed below (a-d). Through systematic examination of each potential variable, multiple modifications were implemented during method development, particularly to improve sensitivity.

a. Contamination: Initial injections of neat standards prepared in 50% acetonitrile were linear from 0.5 - 1000 ng/dL, but the low end of the curve was intermittently visible due to high background. Additionally, the baseline of neat injections was increasing over time. We suspected that either testosterone or another analyte with a similar ionization pattern was accumulating on the column or system.
b. Calibration preparation: The calibrators prepared in the candidate matrix generated biased results (>30%) relative to an outside reference lab. The sample comparison showed no bias (≤-5%) with a set of commercial calibrators.
c. Mobile phase modifier: Testosterone areas were higher with more labor-intensive sample preparations such as SPE and LLE, compared to protein precipitation methods. To glean the benefits of LLE without the partitioning time and supernatant transfer we opted for a SLE sample preparation. The cleanliness of the extracts improved the signal to noise ratio of our LLOQ (1 ng/dL), but the absolute area of the quantification peak was still lower than desired (<10,000).
d. Signal loss after sample extraction in plates: The SLE extraction procedure was optimized in cartridges but was switched to plates to improve throughput. A significant loss (3-fold) in signal was observed when the SLE extraction was switched from cartridges to plates, and the collection from glass vials to plastic plates.

2. Method Information (Optimized)
&bull; Sample Extraction
o Sample volume 200uL
o Supported liquid extraction (SLE)
o Elution solvent: 9:1 Hexane:Ethyl acetate
o Concentrate and reconstitute in 200 uL of 60% methanol
&bull; Instrument Information
o Binary Pumping system
 Multiplex over 2-channels
o MS Triple Quadrupole
&bull; Mobile Phase A: 0.2 mM Ammonium Fluoride in LCMS grade water
&bull; Mobile Phase B: 0.2 mM Ammonium Fluoride in LCMS grade (7:3)(Methanol:Acetonitrile)
&bull; 7-minute gradient, 0.5 mL/min flow rate
o Multiplexing run time ~ 5 minutes
&bull; Column: C18 100x3 mm, 2.6 um with guard cartridge (40°C)
&bull; Injection volume 50 uL
&bull; Quantitative MRM acquisition
&bull; Calibration range 1.0-2000 ng/dL in ultra-low testosterone stripped serum

3. Troubleshooting Steps
a. LC Cleaning/flushing and MS Bakeout
b. Evaluation of mobile phase reagents (i.e., water) and equilibration step
c. Assessment of candidate matrix for calibrators and preparation protocol
d. Comparing different sample preparation and mobile phase additives
e. Evaluation of plastic collection plate vs glass vials

4. Outcomes
a. Contamination:
The LC system was thoroughly cleaned/flushed and the MS was bakeout (Step 3a.) to remove any residual contaminants. To investigate further, we injected blank using a double gradient after a prolonged equilibration period at initial conditions (90% aqueous) in mobile phase A prepared using CLRW. The resulting chromatography showed a noticeable increase in the baseline signal at the analyte retention time. We repeated this step using mobile phase A prepared using bottled DI or LCMS grade water (Step 3b). LCMS grade water effectively reduced the background and risk of contamination but not the DI water.

b. Calibration preparation:
We investigated if the candidate matrix was contributing to the bias. Not surprisingly, several commercial serums and prepared alternatives using BSA and PBS screened were found to contain testosterone. We identified a double stripped ultra-low testosterone serum as a suitable candidate matrix for preparing calibrators (Step 3c.). The bias was corrected only after implementing the following steps to prepare in-house calibrators in double stripped ultra-low testosterone: sonicate the CRM in its ampule, dilute CRM in the same carrier solvent it was shipped in, sonicate and rock the high-level calibrator in matrix before preparing additional calibrators levels.

c. Mobile phase modifier:
Replacing the formic acid mobile phase modifier for a low concentration of ammonium fluoride (Step 3d.) resulted in a 3-fold increase in signal.

d. Signal loss after sample extraction in plates:
Through a series of investigations alternating the sample pre-treatment vessel (plastic vs glass), the SLE format (cartridge vs plate) and collection vessel (plastic vs glass), the root cause of the signal loss was attributed to the plastic collection plate. This was rectified by collecting the samples in glass-coated plates (Step 3e.).

Troubleshooting the Transformation of Arsenic Species in Urine by HPLC-ICP-MS Kathryn Smith (Presenter) ARUP Laboratories

>> POSTER (PDF)

Poster #2a View Map

This poster will be presented and discussed on Wednesday at 18:15 for 15 minutes in Montreal 3 (Track 2).

1. Problem
While preparing a set of four calibrators for six arsenic species in blank human pooled urine, one of the arsenic species (arsenobetaine, AsB) had poor recoveries (0~30%) in calibrators 1-2 along with the presence of an unexpected arsenic peak. Another arsenic species (arsenocholine, AsC) had lower than expected recoveries in calibrator 1-2 (~72%). Only calibrator 4 had acceptable recoveries (within 10% of target) for AsB and AsC. The remaining arsenic species included dimethylarsonic acid (DMA), monomethylarsonic acid (MMA), AsV and AsIII and had an average recovery of 93 ± 3% to target across the four calibrators.

2. Method Information
•50μL urine diluted with 950μL diluent + internal standard (Sb) solution
•Agilent 1260 Infinity II LC
•Agilent 7700 ICP-MS
•PRP-X100 Anion Exchange HPLC column (150x4.6mm), with KrudKatcher Classic HPLC in-Line filter
•MPA – 20mM Ammonium Carbonate + 3% Methanol, pH 8.7
•MPB – 50mM Ammonium Carbonate + 3% Methanol, pH 8.0
•12min analytical step gradient, Flow 1mL/min
•Column oven 20°C
•50μL injection volume

3. Troubleshooting Steps
Three additional lots of blank human pooled urine were spiked with AsB and AsC at a calibrator 1 level (5μg/L) and left on the benchtop overnight. Two of the lots had AsB and AsC recoveries within ±10% of target while the third lot had significant degradation of AsB (<10% recovery) and AsC (80% recovery) along with the presence of the unknown arsenic peak. After a literature search, it has been reported that microorganisms are capable of AsB biotransformation. The four blank pooled urine lots were sent to be cultured and the two lots with stable AsB and AsC recoveries had no growth. The other two lots with AsB and AsC degradation had organisms present and were identified by MALDI-TOF MS as Pseudomonas fluorescens, Serratia species, Proteus vulgaris, and Achromobacter species. These organisms were isolated, inoculated into sterile urine spiked with AsB and AsC each at 5μg/L, and left overnight on the benchtop. Degradation of AsB and AsC (<20% recoveries) along with the appearance of the unknown arsenic species were observed in the Pseudomonas fluorescens and Achromobacter species inoculated urine. When urine was inoculated with Serratia species, there was low AsC recovery (~34%) while the AsB species had enhanced recovery (164%). The Proteus vulgaris and sterile urine (control) had AsB and AsC recoveries within ±10% of target.
4. Outcome
The calibrators were prepared in synthetic urine instead of human pooled urine to avoid preparing calibrators in urine contaminated with bacteria.

Optimized Extraction Protocol for Analysis of 2,3-Dinor 11β-Prostaglandin F2α in Urine Kayla Moehnke (Presenter) Mayo Clinic

Poster #2b View Map

This poster will be presented and discussed on Wednesday at 18:30 for 15 minutes in Montreal 3 (Track 2).

Problem
Historically, the measurement of 2,3-dinor 11β-Prostaglandin F2α (BPG) in urine by liquid chromatography-tandem mass spectrometry (LC-MS/MS) proved challenging due to interferences impacting peak integration, as well as fragment ion ratio discrepancies, both leading to challenges in reporting patient results. Currently, our laboratory method utilizes an ethyl acetate liquid-liquid extraction (LLE), followed by LC-MS/MS. Due to the aforementioned challenges, the opportunity to improve various aspects of the current method by investigating alternative enrichment methods has been identified.
Method Information
• Prepare 1 mL urine sample, calibrator, QC, blank
• Add 25 mcL internal standard, deuterium-labeled 2,3-dinor 11β-Prostaglandin F2α (Cayman Chemical, 9000603)
• Add 50 mcL 50% acetic acid
• Add 1 mL ethyl acetate
• Vortex 1 minute (x3)
• Centrifuge at 3000 rpm for 10 minutes
• Transfer 500 mcL of top ethyl acetate layer to deep well plate
• Dry plate under nitrogen at 50°C for 30 minutes
• Reconstitute with 135 mcL of 40% methanol
Troubleshooting Steps
Optimization of a new BPG sample preparation procedure required several months of experimentation as we strategically evaluated one variable at a time. Some variables were carried through the project to a newly optimized sample preparation procedure, while others were abandoned.
A. A direct comparison of the current ethyl acetate LLE to a solid-phase extraction (SPE) (Agilent, A3967010) was performed, using a protocol developed with a similar analyte, precondition plate, sample transfer, wash, and collect eluate. Initial SPE experiments demonstrated improved signal-to-noise, but interfering peaks continued to be observed.
B. The addition of a third wash step to the SPE protocol was attempted to see if further cleanup would reduce the interference peaks. The initial SPE protocol had two wash steps after the sample transfer; wash #1 500 mcL of water and wash #2 500 mcL of 100% methanol. Variable mixtures and concentrations of methanol, acetic acid, acetonitrile, dichloromethane (DCM), isopropanol, and ammonium hydroxide were evaluated as a wash #3. The addition of a third wash step did not show a significant improvement. For example, observations included low signal-to-noise, poor peak shape, or no signal at all.
C. Modifications to the current wash #2 (methanol) and elution buffer (1% acetic acid in methanol) in the SPE protocol were evaluated to further reduce peak interferences. The optimization of both the wash #2 and elution buffer identified a new combination that improved signal intensity and peak shape. The new combination included a change to 30% methanol for wash #2 and to 1% acetic acid in 75% methanol for the elution buffer. Despite this improvement, some fragment ion ratios continued to give discordant results, including linearity discrepancies.
D. Reversed phase SPE (8E-S100-AGB, Phenomenex) was evaluated, as an alternative chemistry for purification. While using the established SPE protocol, we evaluated methanol, DCM, and isopropanol as options for wash #2. Overall, the reversed phase SPE did not resolve ion ratio discordance and instead appeared to worsen the discrepancies. Therefore, reversed phase SPE was not explored any further.
E. Supported liquid extraction (SLE) was evaluated. Both diatomaceous (Biotage, 820-0400-P01) and synthetic plates (Agilent, 5610-2004) were evaluated using manufacturer-suggested protocols. This included the evaluation of multiple elution buffers such as methyl tert-butyl ether, ethyl acetate, DCM, and n-butyl-chloride. The SLE extractions proved to be less appropriate for BPG extraction. We observed low and variable recovery.
F. Finally, the current extraction methods were directly compared to variations of a SPE method, working through combinations of the most promising wash and elution buffers. The current LLE method was directly compared to SPE, with results favorable for SPE. The SPE method was then compared to a SPE method with DCM as wash #2. Finally, the wash #3 and elution buffer were optimized by comparing, wash #3 (methanol vs 20% methanol + 1% acetic) and the elution buffer (1% acetic acid in methanol vs 1% acetic acid in 80% methanol). Collectively, improved results, regarded as a decrease in interfering peaks, better ion ratio agreement and improved linearity, were observed with the optimized SPE method using methanol for wash #2, 20% methanol+ 1% acetic acid for wash #3, and 1% acetic acid in 80% methanol as the elution buffer.
Outcome
• Prepare 250 mcL urine sample, calibrator, QC
• Add 50 mcL internal standard, deuterium-labeled 2,3-dinor 11β-Prostaglandin F2α
• Add 50 mcL 1N NaOH
• Add 1 mL water
• Precondition SPE plate, with positive pressure manifold
o 500 mcL methanol, ~1 psi
o 500 mcL water, ~1 psi
• Transfer 1250 mcL sample into SPE plate
o 5 psi, 3-5 minutes
• Wash SPE plate
o 500 mcL water, 4 psi, 3 minutes
o 500 mcL methanol, 3 psi, 3 minutes
o 500 mcL 20% methanol + 1% acetic acid, 3 psi, 3 minutes
• Elute with 80% methanol + 1% acetic acid + 1 mcg/mL estriol (x2)
o 100 mcL, <1 psi, 30 seconds
o 100 mcL, <1 psi, 3 minutes
• Add 300 mcL water to eluate
Conclusion
As an inference-prone analyte, BPG proves difficult to isolate from urine samples and accurately measure. Troubleshooting the extraction variables led to the successful development of a SPE method that enriches BPG from urine while minimizing interferences. When isolated, each optimized variable marginally improved assay performance. Synergistically, a combination of the extraction medium and optimized reagent chemistries provided the best chromatographic performance with reduction of interferences, improving linearity and maintaining validation acceptance criteria.

You Don’t Know What You Don’t Know : How Automating Data Review Helped Uncover Chromatography Variations and Issues Emily Chegwidden (Presenter) Cleveland Clinic

Poster #3a View Map

This poster will be presented and discussed on Wednesday at 18:45 for 15 minutes in Montreal 3 (Track 2).

Summary
Through the implementation of automated chromatography review in our clinical laboratory, we identified notable differences in instrument performance affecting chromatography, particularly peak shape (tailing). Prior to this, acceptable tailing criteria were not established for manually reviewed results from the instrument software, and visual assessment of peak tailing was purely subjective. Upon automating review with defined peak tailing criteria, chromatography from one instrument consistently met the assay’s established limits while the other did not for 25-50% of samples per batch. We hypothesized that the differences are the result of an ongoing LC problem that was not identified by manual review of chromatograms. Troubleshooting and service support is ongoing to resolve the underlying issue. Here, we highlight the value of using an automated software for chromatography data review as a tool for early identification of assay performance issues and to guide troubleshooting.

Background
Manual chromatography review is time-intensive and subjective, and it relies heavily on the experience and expertise of the reviewers. Advancements in analytical software have improved the review process by making it easier for clinical laboratories to apply rules or flags to various aspects of chromatographic results. Automation of chromatography review achieves standardization, reduces subjectivity, decreases manual time and labor, and improves data quality and consistency. Standardization of chromatography review is essential in ensuring high quality and timely and accurate reporting of clinical laboratory results.

1.Problem
Automated chromatography review was implemented in our clinical laboratory for the analysis of methylmalonic acid (MMA) in plasma and serum by LC-MS/MS. The MMA assay is validated on two LC-MS/MS instruments. During the development and implementation of automated chromatography review, decision criteria for chromatography flags were initially determined using recommended values and laboratory data collected by the primary LC-MS/MS instrument. While reviewing data and applying preliminary and recommended criteria, we noticed that a significant number of patient samples displayed asymmetrical peak shape (on average 25 – 50% of samples per batch of ~60 samples) and were flagged by the automated review software for chromatography issues (peak tailing). The recommended acceptable ratio for peak tailing is generally ≤ 2.00 (measured at 20% of peak max). Even with a more liberal setting (> 2.30), we found that a number of patient samples (5 or fewer per batch) still flagged for peak tailing. MMA data were then collected and reviewed from the other LC-MS/MS instrument, for which we observed a noticeable improvement of peak symmetry and did not observe flags for peak tailing. Final settings for peak tailing were established based on data review from this other LC-MS/MS instrument (<1.70), and when all MMA results were analyzed using this criteria, we found evidence that one LC-MS/MS instrument consistently produces better chromatography than the other. Under manual review conditions (instrument software and subjective judgment), these differences in chromatography went undetected.

2.Method Information
Analytical Method:
MMA is measured by LC-MS/MS. MMA is extracted from serum or plasma with a deuterated internal standard using solid phase extraction. The eluent is dried down, reconstituted, and injected onto the LC-MS/MS for separation and quantitative SRM acquisition.
a. Instrument(s): Thermo LX-2 Transcend II HPLC and TSQ Quantis MS/MS
b. Column: Restek Force C18, 100 mm x 3.0 mm, 3 µM
c. Mobile Phase A: 0.5% formic acid in water
d. Mobile Phase B: 0.5% formic acid in methanol
e. Flow rate: 0.7 mL/min (95% A: 5% B)
f. Injection volume: 25 µL

Automated Chromatography Review Software System:
Raw data collected by the LC-MS/MS software is transferred to the automated chromatography review software system and undergoes data analysis, quantification, and quality control assessment using custom-built and laboratory defined parameters.
a. Software System: Indigo BioAutomation Ascent v 4.2.0
b. Select rules (flags) with established laboratory criteria for MMA:
i. RT Shift X min > 0.05 min
ii. Peak Tailing at 20% X > 1.70
iii. ISTD Response Recovery X% > 170% or <50%

3.Troubleshooting Steps
We verified that the instrument methods and parameters were identical on both instruments and matched validated settings. We performed duplicate runs on both instruments using the same extracted samples, mobile phases, and analytical column to rule out variation from sample preparation, reagents, or consumables. Manual review of chromatographic results showed slightly more tailing from one instrument; automated chromatography review of these same results clearly demonstrated peak tailing for the same instrument with multiple samples flagged. We used the software to evaluate select historical batches (collected at least 1 month prior or earlier) to determine when chromatography quality began to decline.

Additional on-site troubleshooting has been provided by field service engineers. Troubleshooting measures have focused on the LC and have included, but are not limited to, verification of all connections and fittings and replacement of various consumables (i.e.: inject valve and stator and rotor seal on bypass valve). Using automated chromatography review to evaluate the results obtained throughout the troubleshooting process has demonstrated that there are still chromatography issues with one of the two LC-MS/MS instruments.

4.Outcome
The laboratory is still investigating the source of peak tailing in the MMA assay, including determining when the chromatography quality between the two LC-MS/MS instruments started to diverge. Additional (escalated) analytical troubleshooting is in progress with the technical support team. Currently, all MMA runs are being performed on one single LC-MS/MS.

MALDI-MSI Analysis of Neurosteroids and Neuropeptides in the Human Brain Katrina Edmond (Presenter) University of Wollongong, Australia

Poster #8a View Map

This poster will be attended on Thursday at 09:15 for 1 hour 15 minutes in the Exhibit Hall.

Background:
Transcriptomic analysis alone of steroids and neuropeptides in the human brain is insufficient in allowing us to recapitulate the biological basis for how stress contributes to the development of brain disorders including schizophrenia, major depression and bipolar disorder. A 2023 Nature Biotechnology study by Vicari and colleagues reported a multimodal approach to tissue analysis which combines histology, mass spectrometry and spatial transcriptomics to give a holistic overview of mRNA transcripts, proteins, lipids and low molecular weight metabolites within a single tissue sample (1). If this pipeline could be adapted to detect neurosteroids and neuropeptides, this new workflow could provide critical information about the underlying biological processes which modulate the status of upstream regulation of gene and protein expression by neuropeptides/steroids in pathological conditions, including psychopathology.

Problem:
Limiting factors of steroid and neuropeptide analysis include their low in vivo concentrations (especially when compared to endogenous lipid species), high spatial heterogeneity of expression, and dynamic rates of metabolism/stability in postmortem tissue.

Method Information:
Matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) is an established technique for spatially resolved molecular analysis which we are establishing to examine neuropeptide/neurosteroid levels in the human brain with spatial resolution. Briefly, sequential 10-12μm sections of fresh frozen human brain cortex tissue are sectioned using a cryostat, then mounted onto a 10x Genomics Visium barcoded gene expression array glass microscope slide. Slides are stored at -80oC, then thawed in a desiccator for an hour prior to use. To promote ion formation and protect the tissue from degradation, a MALDI matrix whose composition depends on the target molecular species (e.g., 2,5-dihydroxybenzoic acid for neuropeptides), is applied to the tissue using an automated spraying system. Following matrix application, samples are analysed on a Thermo Fisher Scientific Orbitrap Fusion. Following MALDI-MSI, samples are washed thrice in pre-chilled methanol to remove residual matrix and can then be stored at -80oC until Visium processing.

Troubleshooting Steps:
So far, we have focused our optimisation on neuropeptides, with the primary troubleshooting steps being:
- Tissue preparation. Thickness of sample, storage conditions, thawing methods.
- Pre-matrix washes to reduce the lipid content of the tissue and limit the interference of comparatively strong lipid signal peaks. Currently using 30s in 100% chloroform.
- Concentrations of matrix and its delivery solvent. Currently using 50% acetonitrile, 50% water and 0.2% trifluoracetic acid with between 25-35mg/mL DHB at between 4-6 passes.
- Instrument settings for increased signal strength in the larger m/z peptide ranges (i.e., >1000m/z), currently working through trialling conditions of 400oC tube temperature, 60-120% RF lens, 500-2000 m/z scan rang and 7-12% laser power in positive mode.

Outcome:
While still in the early stages of establishing methods for the analysis of steroids and neuropeptides in the human brain using our Orbitrap Fusion, I am looking forward to feedback on our intended next steps (especially analysis of lowly concentrated neurosteroids) and potentially identifying additional components for optimisation.

References:
(1) Vicari M, Mirzazadeh R, Nilsson A, Shariatgorji R, Bjärterot P, Larsson L, et al. Spatial multimodal analysis of transcriptomes and metabolomes in tissues. Nature Biotechnology. 2023.

Failed Proficiency Result for 11-Nor-9-Carboxy-THC Rene Garay (Presenter) Duke University Health System

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Poster #34b View Map

This poster will be presented and discussed on Wednesday at 19:00 for 15 minutes in Montreal 3 (Track 2).

Title: Failed proficiency result for 11-nor-9-carboxy-THC
1. Problem
Laboratory failed a proficiency sample result for THC-COOH. Investigation was launched to determine root cause of failure.
2. Method Information
• Waters LC-MS system
o Acquity Binary Solvent Manager
o Acquity Sample Manager
o Acquity Column Manager
o Xevo TQ-S micro
• Mobile Phase A: 0.005% Formic Acid in Water
• Mobile Phase B: 0.01% Formic Acid in Acetonitrile
• 7 minute gradient method, 0.4 mL/min
• Column: 2.1 x 100 mm, 1.7 µm C18
• Column Temp: 50°C
• Injection volume: 7.5 µL
• Quantitative MRM acquisition
• UTAK drug free urine spiked with KF
• Cerilliant Certified Reference Material
o Calibrator analyte: (±)-11-nor-9-Carboxy-Δ9-THC (Item # T-006)
o Internal Standard: (±)-11-nor-9-Carboxy-Δ9-THC-d3 (Item # T-004)

3. Troubleshooting Steps
THC-COOH slope drift was observed in calibration as the 5-day work week passed. Protocol at the time was to pull new calibrator set from -80°C freezer on Monday, use throughout the week (interim storage at 4°C refrigerated temperature), and discard on Friday. Slope drift appeared to coincide with days of the week. A downward trend in slope value was determined. Stability of THC-COOH in calibrator solution was questioned. Outlier sample was recalculated using calibration slope from different days of the week. The recalculated results would pass using calibrations from earlier days of the week.

4. Outcome
Calibrator set protocol was changed so that sets would be discarded after use on Wednesday, and a new set would be pulled on Thursday. This set would then be discarded on Friday. Ideally, a new set would be pulled every day, but the current composition of the calibrator set would make it too laborious a task to make daily aliquots. The slope of the calibration curve is monitored daily, and any variation exceeding the cutoff is promptly addressed.