Apr 28, 2026
Vialling of label-free GAG disaccharide Standards and Samples for HILIC-MS/MS analysis
- H Davies-Strickleton1,2,
- James Allsey2,
- Douglas Dyer3,
- David Knight2
- 1Discovery Research Platform, Manchester Cell-Matrix Centre, University of Manchester, UK;
- 2Biological Mass Spectrometry Core Facility, University of Manchester, UK;
- 3Manchester Cell-Matrix Centre, Lydia Becker Institute of Immunology and Inflammation, University of Manchester, UK
- BioMS CRF, UoM
Protocol Citation: H Davies-Strickleton, James Allsey, Douglas Dyer, David Knight 2026. Vialling of label-free GAG disaccharide Standards and Samples for HILIC-MS/MS analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl46progo5/v1
Manuscript citation:
A manuscript of this work is in preparation, which details the method and demonstration of its utility by application to a range of different samples as biological sources.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: December 04, 2025
Last Modified: April 28, 2026
Protocol Integer ID: 234189
Keywords: vialling of gag disaccharide sample, gag disaccharide preparation sample preparation, gag disaccharide preparation sample preparation of label, gag disaccharide sample, gag disaccharide preparation, gag disaccharide standard, equimolar disaccharide standards for injection, equimolar disaccharide standard, free gag disaccharide standard, free gag disaccharides from biological tissue, free gag disaccharide hilic, preparation of equimolar disaccharide stock, gag disaccharide, free gag disaccharide, equimolar disaccharide stock, disaccharide, sample preparation, samples for hilic, biological tissues equimolar stock solution preparation, sample preparation of label, ms analysis from biological tissue, ms analysis this protocol, samples for analysis hilic, step guide for the preparation, ms data acquisition, preparation, ms analysis, sample
Funders Acknowledgements:
Wellcome Trust Discovery Research Platform (HDS, DD)
Grant ID: 226804/Z/22/Z
Wellcome Trust Career Development Award (DD)
Grant ID: 319823/Z/24/Z
Abstract
This protocol provides a detailed step-by-step guide for the preparation and vialling of GAG disaccharide Samples and Equimolar Disaccharide Standards for injection and analysis by HILIC-MS/MS.
This is the fourth protocol in a collection, which documents the details of all steps required for label-free GAG disaccharide HILIC-MS/MS analysis from biological tissues:
- Vialling of Standards and Samples for analysis
Following sample disruption and GAG disaccharides generation (according to protocols 1-2) and the preparation of Equimolar Disaccharide Stocks (protocol 3), Samples and Standards are vialled here. Subsequently analysis can then be performed according to protocol 5 in the collection.
Protocol materials
∆UA-2S GlcNCOEt-6S (I-P) internal standard disaccharideIduronCatalog #HD009
Ammonium acetateVWR International (Avantor)Catalog #84885.180
Acetonitrile LC-MS GradeFisher ScientificCatalog #A/0638/15
Solution preparation
250 mM Ammonium Acetate Stock
Prepare around 1 L, based on the following example:
- Weigh 19.27 g Ammonium acetateVWR International (Avantor)Catalog #84885.180
- Add to 1 L ddH2O
- Mix well
- Store for up to one year at 4 °C
Note
Exact weights and volumes can be adjusted using the formula below.
Weigh approximately 19.27 g ammonium acetate, then use the formula below to calculate the volume of ddH2O to add (V)
V = m / (C*MW)
where:
V = desired volume (L)
m = mass of solute (g)
C = desired concentration (M) = 0.25
MW = molecular weight = 77.08
Example
If 20 g ammonium acetate was weighed then the volume required would be:
20/(0.25*77.08) = 1.037 L
Solution A (77% acetonitrile, 45 mM ammonium acetate)
Note
Solution A is prepared by combining:
- 77 parts acetonitrile
- 18 parts 250 mM Ammonium Acetate Stock (18 in 100 dilution to give final concentration of 45 mM)
- 5 parts water
- Total: 100 parts
An example preparation for 100 mL is given below. Should a higher volume be needed, multiply all component volumes accordingly (e.g. for 200 mL, multiply all the volumes below by 2)
To make 100 mL in a duran bottle, use a measuring cylinder to add:
- 77 mL Acetonitrile LC-MS GradeFisher ScientificCatalog #A/0638/15
- 18 mL 250 mM Ammonium Acetate Stock for stock preparation
- 5 mL ddH2O
- Mix well and allow to reach room temperature
Store at room temperature for up to 3 months.
Note
For both Samples and Standards, a dilution factor (1 in 20) and vial volume (60 μL) were found during method development to provide good intensity by HILIC-MS/MS.
- These parameters should be kept consistent across Samples and Standards in an analytical run.
- For new applications, such as ultra-low starting amounts of material, these parameters could be tested and explored further, whilst being consistent across both samples and standards.
-----------------------------------------------------------------------------------------------------
Follow the step cases below for:
- Equimolar Disaccharide Standard preparation and vialling
- Sample dilution and vialling
Standard Dilution and Vialling9 steps
Equimolar Disaccharide Standard Preparation and Vialling
Note
Here, these are diluted 1 in 20 in Standard Diluent to prepare Equimolar Disaccharide Standards for subsequent HILIC-MS/MS analysis.
-----------------------------------------------------------------------------------------------------
Rationale of 50 nM Equimolar Disaccharide Standard preparation
In this section, an 50 nM Equimolar Disaccharide Standard (a mixture of disaccharide standards at equal molar concentration) is prepared for HILIC-MS/MS injection and analysis.
The purpose of this 50 nM Equimolar Disaccharide Standard is to correct for disaccharide specific variability in analysis and to provide a single point calibration for the approximation of disaccharide amount (semi-quantification).
The semi-quantification presented here is appropriate for the relative quantification of disaccharide amounts, total GAG amounts (by summation of disaccharide amounts) and disaccharide composition (relative ratio of disaccharides compared to total GAG).
(For comparison of relative amounts of disaccharides in samples, without disaccharide composition, standard preparation may not be required.)
Rationale of 5, 500 and 5000 nM Equimolar Disaccharide Linearity Standard preparation
Optional 5, 500 and 5000 nM Equimolar Disaccharide Linearity Standards may also be prepared. These serve as (a) test injections to optimise LC-MS conditions prior to analysis and/or (b) to confirm analyte linearity.
Rationale of Internal Standard addition to Standard Diluent
The Equimolar Disaccharide Standard is spiked with an Internal Standard, to correct for variability in instrument performance.
In a separate protocol for preparation of GAG disaccharides from biological tissues, samples were also spiked with the Internal Standard.
Internal Standard
Note
The internal standard solutions may have been prepared during the sample preparation protocol, and may be used within one week of prep if kept at4 °C .
The internal standard stock is stored at -20 oC in 10 µL aliquots of 1 mg/mL in ddH2O.
Thaw the 1 mg/mL Internal Standard Stock∆UA-2S GlcNCOEt-6S (I-P) internal standard disaccharideIduronCatalog #HD009
Dilute the Internal Standard Stock to make a 10 μg/mL Internal Standard Solution (1 in 100 dilution):
- To the 10 µL aliquot add 990 µL ddH2O and mix well
Dilute the 10 μg/mL Internal Standard Solution to make 1 μg/mL Internal Standard Solution (1 in 10 dilution):
- To a fresh microcentrifuge tube add 100 µL 10 μg/mL Internal Standard Solution
- Add 900 µL ddH2O
- Mix well
- Store at4 °C when not in use.
Standard Diluent (93 parts Solution A; 2 parts 1 μg/mL internal standard)
Note
This Standard Diluent is used to prepare all Equimolar Disaccharide Standards.
It contains high % organic solvent (suitable to HILIC-MS/MS analysis) and the spiked internal standard‡:
- 93 parts Solution A for preparation
- 2 parts 1 μg/mL Internal Standard Solution (in water)
‡The % organic solvent for Standards is the same as that used to prepare Samples. The Standard Diluent includes the addition of Internal Standard, which is already present in Samples due to addition during their preparation. The Internal Standard concentration will be consistent between Sample and Standard vials.
-----------------------------------------------------------------------------------------------------
Prepare just before use and use the same day.
Prepare one Standard Diluent for all Standards.
The volume prepared here is enough for 15 Standards.
- Should a higher volume be needed (e.g. to prepare 30 Standards), multiply all component volumes accordingly (e.g. for 30 Standards, multiply all the volumes by 2)
Using a pipette prepare950 µL Standard Diluent in a glass HPLC vial:
- 930 µL Solution A for solution preparation (note: for organic solvents equilibrate the pipette tip first by fully withdrawing the liquid slowly up and down)
- 20 µL Internal Standard Solution 1 μg/mL for Internal Standard Solution preparation
- Mix well
Prepare Equimolar Disaccharide Standards
Note
Here, these are diluted 1 in 20 in Standard Diluent to prepare Equimolar Disaccharide Standards for subsequent HILIC-MS/MS analysis:
- 50 nM Equimolar Disaccharide Standard for the approximation of disaccharide amount (semi-quantification).
- 5, 500 and 5000 nM are optional Equimolar Disaccharide Linearity Standards to confirm/assess linear response of analytes
-----------------------------------------------------------------------------------------------------
Use a fixed volume of 60 μL for dilutions:
- 57
μL Standard Diluent - 3 μL Equimolar Stock Solution
The following table demonstrates each Equimolar Stock Solution and the Equimolar Disaccharide Standards obtained after dilution:
| A | B | C | D | E | |
| Equimolar Stock Solution (uM) | Equimolar Disaccharide Standard (nM) | Use in HILIC-MS/MS method | Volume of Equimolar Stock Solution to add (uL) | Volume of Standard Diluent to add (uL) | |
| 100 | 5000 | Linearity | 3 | 57 | |
| 10 | 500 | Linearity | 3 | 57 | |
| 1 | 50 | Quantification | 3 | 57 | |
| 0.1 | 5 | Linearity | 3 | 57 |
The concentration of internal standard in each Standard vial is 20 ng/mL. The same concentration is present in the Sample vials.
Equipment
Certified QSertVial™ (vial with fused-in insert) kit, screw thread, 12 x 32 mm, 0.3 mL
NAME
HPLC vial
TYPE
Supelco
BRAND
29391-U
SKU
Prepare each concentration of Equimolar Disaccharide Standard:
Each row in the table above demonstrates an Equimolar Disaccharide Standard. For each row, prepare the Equimolar Disaccharide Standard as follows:
- Label a HPLC insert vial according to column B
- Add 57 µL Standard Diluent (note: for organic solvents equilibrate the pipette tip first by fully withdrawing the liquid slowly up and down)
- Add 3 µL Equimolar Stock Solution (from column A)
- Mix by pipetting
For example for the preparation of the 50 nM Equimolar Disaccharide Standard (row 4 of the table above):
- Add 57 µL Standard Diluent (note: for organic solvents equilibrate the pipette tip first by fully withdrawing the liquid slowly up and down)
- Add 3 µL 1 micromolar (µM) Equimolar Stock Solution
- Mix by pipetting
Prepare Blank Diluent and Blank Internal Standard vials
Prepare Blank Diluent as follows:
- In a HPLC insert vial, add 60 µL Solution A ( )
Prepare blank Internal Standard as follows:
- In a HPLC insert vial, add 57 µL Standard Diluent (note: for organic solvents equilibrate the pipette tip first by fully withdrawing the liquid slowly up and down)
- Then add 3 µL water
- Mix by pipetting
Put sample vials in autosampler 90 minutes before injection to equilibrate to autosampler temperature.
- Blank Diluent
- Blank Internal Standard
- Equimolar Disaccharide Standard: 50 nM
- Optional Equimolar Disaccharide Linearity Standards: 5, 500 and 5000 nM