Liquid chromatography-mass spectrometry method for isomer separation and detection of sugars, phophorylated sugars and organic acids

CE: collision energy * CXP: collision cell exit potential

• ddH₂O: double-distilled water
• DP: declustering potential
• EP: entrance potential
• FW: fresh weight
• HILIC: hydrophilic interaction liquid chromatography
• HPLC: high-performance liquid chromatography
• ID: isotopologue distribution
• LC-MS: liquid chromatography-mass spectrometry
• MRM: multiple reaction monitoring
• PES: Polyethersulfone
• PIPES: Piperazine-N,N′-bis(2-ethanesulfonic acid)
• PVDF: Polyvinylidene fluoride

Vivaclear Mini 0.8 µm PES clarifying filters (Sartorius #VK01P042)* Durapore membrane filter, PVDF hydrophilic, 0.22 µm , 47 mm (EMD Millipore #GVWP04700)

• HPLC pump: Shimadzu Prominence-xR UFLC system
• HPLC column: Intrada Organic Acid (150 mm x 2 mm , 3 µm )
• MS detector: Sciex QTRAP 6500 triple quadrupole-linear ion trap MS with a Turbo VTM electrospray ionization source

A 500micromolar (µM) stock of alpha-ketoglutarate, 2-phosphoglycolate, 2-phosphoglyceric acid, 3-phosphoglyceric acid, 6-phosphogluconate, acetyl-CoA, adenosine diphosphate-glucose, aspartate, dihydroxyacetone phosphate, erythrose 4-phosphate, fructose, fructose 1,6-bisphosphate, glucose 1-phosphate, glucose 6-phosphate, glyceraldehyde phosphate, glucose, glutamine, glutamate, glycerate, malate, phosphoenolpyruvate, piperazine-N,N′-bis(2-ethanesulfonic acid), raffinose, ribitol, ribulose-1,5-bisphosphate, ribulose 5-phosphate, sedoheptulose 7-phosphate, succinate, sucrose, and uridine diphosphate-glucose standards was prepared in ddH₂O containing 0.2% (v/v) formic acid.

Prepare a 250micromolar (µM) working stock in ddH₂O containing 50% (v/v) methanol and 0.2% (v/v) formic acid.

Filter the 250micromolar (µM) stock through a 0.8 µm PES clarifying filter at 2000x g,4°C,0h 0m 0s for 0h 5m 0s.

Serially dilute the filtered 250micromolar (µM) stock in a filtered ddH₂O containing 50% (v/v) methanol and 0.2% (v/v) formic acid to create a standard curve containing 250, 125, 62.5, 31.25, 15.625, 7.813, 3.906, 1.953, 0.977, 0.488, 0.244, and 0.122 µM standards.

Transfer 50µL of each standard concentration into separate 300µL LC vials with inserts.

These internal standards are used for calculation of extraction efficiencies (PIPES for organic acids, ribitol for sugars and phosphorylated sugars, and norvaline for amino acids).

Prepare separate 1mg/mL stocks for each internal standard in 1mL chilled ddH₂O.

For 1mL of 100μg/mL standard mix, mix 100µL of each 1mg/mL stock with 700µL chilled ddH₂O.

Add 15µL 100μg/mL PIPES-Ribitol-Norvaline as internal standards during sample extraction. Polar metabolites are extracted using the protocol adapted from Ma et al., 2017, with only a single water extraction step performed and samples being resuspended in 50µL ddH₂O containing 50% (v/v) methanol and 0.2% (v/v) formic acid and subsequently filtered through 0.8 µm PES clarifying filters at 2000x g,4°C,0h 0m 0s for 0h 5m 0s.

Solvent A: ddH₂O containing 100millimolar (mM) ammonium formate and 10% (v/v) acetonitrile * Solvent B: ddH₂O containing 1% (v/v) formic acid and 75% (v/v) acetonitrile

• Seal wash: ddH₂O containing 20% (v/v) methanol and 0.5% (v/v) formic acid
• Autosampler wash 1: ddH₂O containing 25% (v/v) methanol
• Autosampler wash 2: ddH₂O containing 75% (v/v) methanol
• Vacuum filter buffers using 0.22 µm PVDF hydrophilic membrane filters into clean bottles.

Table 1. HPLC mobile phase gradient

A	B	C	D
Time (min)	Flow (mL/min)	%A	%B
0	0.225	0	100
1	0.225	0	100
5	0.225	12	88
7	0.225	12	88
8	0.225	16	84
10	0.225	16	84
13	0.225	25	75
15	0.225	100	0
19.5	0.25	100	0
20	0.25	0	100
25	0.25	0	100

• All samples are diluted in ddH₂O containing 50% (v/v) methanol with 0.2% (v/v) formic acid.

• A 3µL injection volume is used, the sample tray in autosampler is held at 4°C, and the column temperature is held at 40°C.

Source temperature: 450°C* Ion spray voltage: -4500 V

• Curtain gas: 30 psi
• Ion source gas 1: 30 psi
• Ion source gas 2: 35 psi

Table 2. LC-MS parameters for each compound

A	B	C	D	E	F	G
Q1	Q3	Compound ID	DP (v)	CE (v)	CXP (v)	EP (v)
145	101	Alpha-ketoglutarate	-5	-12	-11	-10
155	79	2-phosphoglycolate	-70	-20	-12	-10
275	79	6-phosphogluconate	-65	-22	-13	-10
808	408	Acetyl-CoA	-35	-46	-23	-10
588	346.1	Adenosine diphosphate-glucose	-30	-32	-21	-10
132	88	Aspartate	-20	-16	-43	-10
199	97	Erythrose 4-phosphate	-5	-32	-11	-10
339	97	Fructose 1,6-bisphosphate	-60	-24	-11	-10
145.1	108.9	Glutamine	-30	-18	-5	-10
146.05	102	Glutamate	-16	-15	-8	-10
179	89	Hexoses	-25	-10	-11	-10
259	79	Hexose phosphates	-10	-28	-7	-10
133	115	Malate	-40	-20	-3	-10
229	79	Pentose 5-phosphates	-25	-60	-15	-10
167	79	Phosphoenolpyruvate	-5	-14	-9	-10
185	79	Phosphoglyceric acids	-30	-20	-11	-10
301	193	PIPES	-35	-34	-31	-10
87	43	Pyruvate	-10	-15	-3	-10
151	89	Ribitol	-50	-16	-9	-10
309	79	Ribulose 1,5-bisphosphate	-55	-70	-9	-10
289	79	Sedoheptulose 7-phosphate	-60	-58	-9	-10
104.001	74	Serine	-16	-15	-8	-10
117	73	Succinate	-5	-16	-7	-10
341	179	Sucrose	-110	-18	-13	-10
169	97	Triose phosphates	-10	-12	-11	-10
565	323.1	Uridine diphosphate-glucose	-125	-32	-2	-10

All analytes are measured in negative ionization mode, with ions being detected using a targeted MRM approach.

Run a full set of all 12 concentrations of the external standard mix on the LC-MS three times total, at the start, middle, and end of sample runs.

Integrate peak areas for each standard peak using the quantitation wizard tool in the Analyst instrument control and data processing software (v.1.6.2) and export the data to Excel.

In Excel, convert peak area units from µM to µmols of compound by multiplying by sample volume (50µL).

Plot the peak areas vs the standard concentrations and fit a linear regression to the data.

For sample runs, compare the measured amounts of PIPES, ribitol, and norvaline internal standards with the known amounts added during sample preparation to calculate the percent metabolite recovery, using PIPES for organic acids, norvaline for amino acids, and ribitol for sugars, nucleotide sugars, and phosphorylated sugars.

For each metabolite, correct for metabolite loss during extraction using the relevant factor.

For each metabolite, calculate the concentration from the sample by solving for x using the linear regression equation of that metabolite’s standard curve.

For each metabolite, determine the lowest concentration on the standard curve that still showed a change in peak area. Calculate the standard deviation of this concentration (SD_low) for all three injections.

Calculate the limits of detection and quantitation for each metabolite by multiplying this standard deviation by 3 or 10 and dividing by the slope of the standard curve.

For sample runs investigating ¹³C-tracer incorporation, the MRM list for compounds of interest was expanded with the set of possible labeled carbon isotopologues in the Q1 ion and the Q3 fragment (if applicable). Since the Q3 fragment for a compound like 3-PGA does not contain any carbons, an increase in label is only possible in the Q1 ion.

Table 3. Monitored isotopologue distribution (ID) for select metabolites

A	B	C
Q1	Q3	Compound ID (13C in Q1/13C in Q3)
133	115	MAL-0/0
134	116	MAL-1/1
135	117	MAL-2/2
136	118	MAL-3/3
137	119	MAL-4/4
185	79	PGA-0/0
186	79	PGA-1/0
187	79	PGA-2/0
188	79	PGA-3/0

Once runs are completed, extract the peak intensities (or areas) of the set of isotopologues for each compound.

To determine isotopologue distribution (ID) of a particular isotopologue (M_i), divide the abundance (A) of that isotopologue by the sum of all isotopologue abundances for that compound.

Once all of the isotopologue distributions have been determined, the average ¹³C-enrichment for each compound can be calculated by determining the sum of each isotopologue multiplied by the number of labeled carbons present and dividing by total number of carbons in that compound.