Valine Analysis Service - Creative Proteomics

Q: Do samples need to be fasted for valine measurement?

Fasting is recommended because BCAAs (including valine) rise post-prandially and can remain elevated for hours, which biases baseline comparisons.

Q: How long can plasma/serum stay on ice before freezing without compromising metabolomics readouts?

Keep blood derivatives cold and limit pre-freeze handling; harmonized SOPs suggest holding on ice up to ~2 hours before snap-freezing as a practical upper bound for robust LC-MS metabolomics.

Q: Are freeze–thaw cycles a concern for amino-acid quantification?

Yes—pre-analytical variation (including repeated freeze–thaw) measurably perturbs metabolite levels, so cycles should be minimized and documented.

Q: What confirms identity in LC-MS/MS beyond retention time?

Dual-transition MRM with ion-ratio agreement and RT windows provides confirmatory evidence; ion-ratio tolerances are typically set within a defined band and justified per matrix/method.

Q: Which criteria define an acceptable LLOQ for valine?

Regulatory method-validation guidance requires adequate accuracy, precision, and signal-to-noise at LLOQ with matrix-matched calibration and QC verification.

Q: How should urine valine (or related targets) be normalized?

Creatinine (and, in some studies, osmolality) normalization is commonly used to correct for urine dilution before statistical analysis.

Q: Is there diurnal variation to consider, or are diet effects dominant?

Dietary intake exerts strong short-term effects on circulating BCAAs; studies of daily rhythmicity show mixed findings, so standardizing feeding status is the safer control.

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What Is Valine—and Why Measure It?

Valine is an essential branched-chain amino acid (BCAA) that supports protein synthesis, nitrogen balance, and energy production—especially in muscle and rapidly dividing cells. Because Val concentrations shift with diet, training, metabolic stress, and drug exposure, quantitative LC-MS/MS provides the reproducible readouts needed to compare conditions, map pathway changes, and link phenotype to mechanism. Co-measurement with leucine/isoleucine and context markers (alanine, glutamine/glutamate) strengthens interpretation of BCAA utilization and central-carbon metabolism across plasma/serum, CSF, urine, tissues, and cell systems.

Valine Analysis Services by Creative Proteomics

At Creative Proteomics, we offer a comprehensive suite of LC‑MS/MS services centered on valine quantification, designed for scientists aiming to decipher BCAA metabolism and related pathways.

Valine absolute quantification in plasma, serum, CSF, urine, tissue lysates, and cell culture systems
BCAA panel analysis: Valine, Leucine, and Isoleucine co-quantified to evaluate metabolic status
Optional analyte extensions: Alanine (transamination), Glutamine/Glutamate (nitrogen turnover), Phenylalanine/Tyrosine (aromatic amino-acid pathways)
Custom ratio outputs: BCAA ratios, optional Phe/Tyr ratio when co-measured
Flux tracing capability: ¹³C/¹⁵N-labeled Valine with full isotopologue readout for pathway flux studies

Why Choose Our Valine Analysis Service?

Broad Dynamic Range – Quantitative range validated over 0.5–500 µM with excellent linearity
High Sensitivity – Matrix-specific LLOQ as low as 0.5 µM, with S/N ≥10 at lowest level
Strong Linearity – Weighted (1/x²) regression with R² ≥ 0.995 across all batches
Precise & Reproducible – Intra-batch %RSD ≤ 10%, inter-batch ≤ 15% at QC levels
Accurate Quantification – Back-calculated %RE within ±10% for calibrators and controls
Selectivity Assured – Dual MRM transitions with ion-ratio deviation <25% and strict RT matching
No Carryover – Verified in post-high blanks, signal <0.1× LLOQ
Panel-Compatible – Co-analysis with BCAAs (Leu/Ile) or amino acids (Glu, Gln, Ala, Phe, Tyr) without compromising performance
Flexible Throughput – Runtime per sample <8 minutes, adaptable for targeted studies or screening

Valine LC‑MS/MS Method and Analytical Performance

Platform: Agilent 1260 Infinity II HPLC + 6495C Triple Quadrupole LC-MS/MS (MRM)

Chromatography: HILIC (amide) or reversed-phase (by matrix)

Internal Standard: isotope-labelled Val (preferred) or validated analogue

Preparation: cold protein precipitation ± derivatization (if justified by matrix/sensitivity); low-temperature handling to minimize conversion

Acquisition: ESI(+) MRM; example transitions Val m/z 118.1→72.1 (quant) and 118.1→55.1 (qual); IS matched transitions

Processing: MassHunter or an equivalently validated pipeline (ion-ratio check, RT window, peak integration review)

Agilent 6495C Triple Quadrupole (Figure from Agilent)

Agilent 1260 Infinity II HPLC (Fig from Agilent)

Valine Quantification Workflow: From Sample to Report

Sample Intake

Sample type, volume, and storage are verified; deviations are documented before processing.

Preparation

Protein precipitation and matrix-optimized extraction under cold conditions to preserve valine stability.

LC-MS/MS Acquisition

HILIC or reversed-phase LC coupled with triple quadrupole MS in MRM mode; dual transitions per analyte.

Quantification & QC

Matrix-matched calibration with isotope-labeled internal standard; multi-level QC ensures accuracy and reproducibility.

Reporting

Results delivered as concentration tables with optional BCAA ratios, chromatograms, and QC summaries.

Sample Requirements for Valine Testing

Matrix	Minimum amount	Container	Pre-analytical handling	Storage & shipping	Notes / Normalization
Plasma / Serum	≥ 80 µL (ideal 100 µL)	Low-bind PP microtube; EDTA or heparin for plasma	Keep on ice; prompt centrifugation; avoid hemolysis	−80 °C; ship on dry ice	Record anticoagulant; limit freeze–thaw (≤3)
CSF	≥ 60 µL	Sterile, low-bind tube	Handle cold; minimize dwell time	−80 °C; dry ice	No additives
Urine	≥ 250 µL	PP tube	Mix gently; note dilution or SG if applicable	≤ −20 °C (short) or −80 °C; cold ship	Optional normalization: creatinine / osmolality
Tissue homogenate	≥ 25 mg eq.	Pre-chilled, low-bind tube	Rapid quench/extraction under cold conditions	−80 °C; dry ice	Provide wet weight & buffer details
Cell lysate / pellet	≥ 50 µL lysate or ≥ 0.5–1×10⁶ cells	Low-bind tube	Work cold; clarify as needed	−80 °C; dry ice	Report protein concentration or cell count
Cell culture supernatant / media	≥ 250 µL	Sterile PP tube	Quench promptly; remove debris by spin	−80 °C; dry ice	Note timepoint, cell density, treatment

General tips: aliquot to avoid repeat freeze–thaw; include a manifest (sample ID, matrix, volume/mass, collection time, treatment, dilution/normalization plan).

What You Receive: Deliverables from Our Valine Analysis

Concentration tables (SI units): Valine results per sample/matrix with sample IDs and any dilution factors.
Calibration summary: Fit model (e.g., 1/x²), range, R², and back-calculated accuracy.
QC overview: Precision/accuracy at L/M/H levels with pass/fail notes.
Representative MRM chromatograms: Quantifier/qualifier traces with RT window and ion-ratio check.
Data files: PDF report plus CSV/Excel; raw files available on request.
If co-measured: BCAA ratios or study-specific indices included.

Valine calibration curve with residual error plot showing high linearity and accuracy.

Weighted (1/x²) linear regression of valine (0.5–500 µM) showing R² = 0.998 and residual errors within ±10% across all calibrators.

Valine MRM chromatograms comparing blank, LLOQ, and real plasma sample with labeled RT and S/N.

MRM chromatograms for valine in blank plasma (A), LLOQ 1.0 µM (B), and real sample (C). Clear RT alignment and ion-ratio confirmation demonstrate method specificity and sensitivity.

Applications of Valine Analysis in Research and Industry

Metabolic Research

Track BCAA metabolism and assess shifts in nutrient utilization.

Nutritional Studies

Evaluate valine levels in response to dietary interventions or fasting.

Cell Culture Monitoring

Quantify valine uptake/release to optimize media and growth conditions.

Exercise Physiology

Monitor valine turnover in muscle metabolism during training studies.

Fluxomics

Use isotope-labeled valine to trace catabolic routes and carbon flow.

Amino Acid Panel Profiling

Include valine in multiplexed amino acid assays for system-wide insights.

Do samples need to be fasted for valine measurement?

Fasting is recommended because BCAAs (including valine) rise post-prandially and can remain elevated for hours, which biases baseline comparisons.

How long can plasma/serum stay on ice before freezing without compromising metabolomics readouts?

Keep blood derivatives cold and limit pre-freeze handling; harmonized SOPs suggest holding on ice up to ~2 hours before snap-freezing as a practical upper bound for robust LC-MS metabolomics.

Are freeze–thaw cycles a concern for amino-acid quantification?

Yes—pre-analytical variation (including repeated freeze–thaw) measurably perturbs metabolite levels, so cycles should be minimized and documented.

What confirms identity in LC-MS/MS beyond retention time?

Dual-transition MRM with ion-ratio agreement and RT windows provides confirmatory evidence; ion-ratio tolerances are typically set within a defined band and justified per matrix/method.

Which criteria define an acceptable LLOQ for valine?

Regulatory method-validation guidance requires adequate accuracy, precision, and signal-to-noise at LLOQ with matrix-matched calibration and QC verification.

How should urine valine (or related targets) be normalized?

Creatinine (and, in some studies, osmolality) normalization is commonly used to correct for urine dilution before statistical analysis.

Is there diurnal variation to consider, or are diet effects dominant?

Dietary intake exerts strong short-term effects on circulating BCAAs; studies of daily rhythmicity show mixed findings, so standardizing feeding status is the safer control.

The Brain Metabolome Is Modified by Obesity in a Sex-Dependent Manner

Norman, J. E., Milenkovic, D., Nuthikattu, S., & Villablanca, A. C.

International Journal of Molecular Sciences

Year: 2024

Characterization of Dnajc12 knockout mice, a model of hypodopaminergia

Deng, I. B., Follet, J., Fox, J. D., & Farrer, M. J.

bioRxiv

Year: 2024

Untargeted metabolomics reveal sex-specific and non-specific redox-modulating metabolites in kidneys following binge drinking

Rafferty, D., de Carvalho, L. M., Sutter, M., Heneghan, K., Nelson, V., Leitner, M., ... & Puthanveetil, P.

Redox Experimental Medicine

Year: 2023