TR09A

Organic Acid Analysis Column for LC-MS

Organic acid LC-MS analysis without any derivatization
Ideal for carboxylic acids and other anions
Full general organic acid screens within 10min
One-minute analyses possible on high-throughput columns
3µm spherical pure silica / specialized stationary phase for organic acid (IEX mode, NP mode)

Total organic acid LC-MS analysis for TCA cycle

oxaloacetic acidaconitic acidfumaric acidpyruvic acidcitric acidisocitric acidmalic acidsuccinic acid123456 min

The di-, and tri- organic acids of the TCA cycle have historically been difficult for thefollowing reasons:High polarity makes them difficult to retain on traditional reversed-phase columns
Multivalent acids containing several different anionic pKa groups, typically show poor peak shape
Direct detection on UV is difficult
Conductivity detection is not compatible with ion-exchange modes under gradient elution
Pre/post- derivatization methods require a complicated HPLC system

We have developed an innovative HPLC column for total organic acid analysis using
LCMS. This column does not require any derivatization method protocols, and almost allorganic acids can be analyzed within 10 min.
We do not guarantee this column will be suitable for all organic acids. Zwitter ions such as amino acids or peptides are not applicable.

Imtakt USA (info@imtaktusa.com)
North America

Imtakt Corp./JAPAN (info@imtakt.com)
Other Countries

TR09AE-SG31

10min LC-MS analysis of conventional organic acids
Intrada Organic Acid was designed for LC-MS analysis of a wide range of organic acids. It is suitable formost anionic structures ranging from weak to strong anions. Additionally, nearly all organic acids can beanalyzed within 10 minutes.

General organic acid LC-MS analysis (IEX mode)[M-H]- m/z = 253.0

pyromellitic acido-phthalic acidlevulinic acidtrimesic acid

[M-H]- m/z = 165.0[M-H]- m/z = 115.0[M-H]- m/z = 209.0[M+HCOO]- m/z = 133.0

n-butyric acid (2mg/mL)
cis-aconitic acidglutaric acidfumaric acidlactic acidtrans-aconitic acidacetic acid (5mg/mL)
gluconic acidbutane-1,2,3,4-tetracarboxylic acidphosphoenolpyruvic acid
2-hydroxyisobutyric acidketoglutaric acid
2-keto-D-gluconic acid
2-hydroxy-3-methylbutyric acid

[M-H]- m/z = 173.0[M-H]- m/z = 131.0[M-H]- m/z = 115.0[M-H]- m/z = 89.0
[M-H]- m/z = 173.0[M+HCOO]- m/z = 105.0[M-H]- m/z = 195.0[M-H]- m/z = 233.0[M-H]- m/z = 167.0[M-H]- m/z = 103.0[M-H]- m/z = 145.0[M-H]- m/z = 193.0[M-H]- m/z = 117.0[M+HCOO]- m/z = 119.0

propionic acid (1mg/mL)
glycolic acidmaleic acidglucose-6-phosphate
pyroglutamic acid
oxaloacetic acid

[M-H]- m/z = 75.0
[M-H]- m/z = 115.0[M-H]- m/z = 259.0[M-H]- m/z = 128.0[M-H]- m/z = 131.0[M-H]- m/z = 161.03-hydroxy-3-methylglutaric acidN-glycolylneuraminic acidadipic acidglucose-1-phosphaten-valeric acid (1mg/mL)
benzoic acidN-acetylneuraminic acidpyruvic acid

[M-H]- m/z = 324.1[M-H]- m/z = 145.0[M-H]- m/z = 259.0[M+HCOO]- m/z = 147.0[M-H]- m/z = 121.0[M-H]- m/z = 308.1[M-H]- m/z = 133.0[M+HCOO]- m/z = 133.0

isobutyric acid (1mg/mL)
glyoxylic acidmesaconic aciditaconic acidL-tartaric acid
3-hydroxypropionic acidcitric acid

[M-H]- m/z = 73.0
[M-H]- m/z = 129.0[M-H]- m/z = 129.0[M-H]- m/z = 149.0[M-H]- m/z = 89.0
[M-H]- m/z = 191.0[M-H]- m/z = 145.0

3-methylglutaric acidisocitric acid
isovaleric acid (1mg/mL)
malic acidsuccinic acid

[M-H]- m/z = 191.0[M+HCOO]- m/z = 147.0[M-H]- m/z = 133.0[M-H]- m/z = 117.0012345678910 minA07

Intrada Organic Acid, 150 x 2 mm
A: acetonitrile / water / formic acid = 10 / 90 / 0.1
B: acetonitrile / 100mM ammonium formate = 10 / 90
General conditions (IEX Mode) 0%B (0-1min), 0-100%B (1-7min), 100%B (7-10min)
0.2mL/min, 37°C, MS (ESI, SIM, Negative)
5µL (100µg/mL in 0.1% HCOOH)
Organic acids come in a wide variety of structures. For example, there are mono, di, tri, hydroxy, keto, aromatic, sulfonic, sulfuric andphosphoric acid types. It is quite difficult to analyze all of these compounds with a single stationary phase, until now.
The Intrada Organic Acid column was designed with this goal in mind, to be able to analyze nearly all organic acids on one column. It
was developed to separate these compounds by recognizing individual anionicity, and polarity, using a unique stationary phase
optimized for the ideal adsorption and desorption for the widest possible range of acidic structures, all while maintaining LC-MScompatibility. Intrada Organic Acid column is the world first silica-based LC-MS column for direct organic acid analysis without theneed for any type of derivatization.

2

Analysis of various organic acid structures
Polyvalent acid and polycarboxylic acid LC-MS analysis (Normal-phase mode)
HOOC

COOH

HOOC

COOH

While the Intrada Organic Acid column has been primarily designed forweak acids, polyvalent or polycarboxylic acids can also be analyzed. It may
be difficult to elute them on this IEX column, however, due to their strong
ionic interaction. In order to resolve this issue, additional technology wasbuilt into the stationary phase structure, which utilizes normal-phase mode
under neutral pH conditions. This highlights another advantage of thedesign of this silica-based column over traditional polymer-based IEX
columns, because changes in the organic composition are possible. This
allows for a greater degree of optimization of the mobile phase for a widevariety of needs.
There are occasions where using the Intrada Organic Acid column undernormal-phase conditions is preferred.

pyromellitic acid(0.94µg/mL)[M-H]- m/z = 253.0

COOH
HOOC

COOH

HOOC

COOH
COOH

[M-H]- m/z = 341.0

mellitic acid (25µg/mL)

General conditions (Normal-phase mode)OOHOPHOPOHOOOHOO POHOHO[M-H]- m/z = 658.91OHO

POH0OHOHOPOO2Pphytic acidOHOH(250µg/mL)

O345678910 min
A01

Intrada Organic Acid, 150 x 2 mm
A: acetonitrile / 100mM ammonium acetate = 95 / 5
B: acetonitrile / 100mM ammonium acetate = 10 / 90
0%B (0-1min), 0-100%B (1-4min), 100%B (4-10min)
0.2 mL/min, 37 °C, MS (ESI, SIM, Negative)
5 µL (50mM AcONH4 solution)

Analysis of various organic acid structures using General Conditions (IEX mode)
Sulfonic acidSulfuric acid

H3C

[M-H]- m/z=171.0[M-H]- m/z=157.0

HOCH SO 2[M-H]- m/z=111.0

3CH3SO3-[M-H]- m/z=95.0[M-H]- m/z=97.0

compounds arepervasive in the life sciences
SO42and are notoriously difficult
to analyze due to their low
retention on reversed-phasesodium methanesulfonate
columns. Using this column,
sodium sulfate however, we can analyze
thesecompounds
with
S2O32extremely short run-times.

012

Nucleotide
Phosphoric Acid
phosphoric acidOHOP OHOH

[2M-H] m/z = 195.0
NH2NNsodium hydroxymethanesulfonate Phosphate

NN
OO
CH2OO

OATPP O P O P OHOHOHOH[M-H] m/z = 506.0
NH2NADP

NNNOO
CH2OO

P O P OH
OH

OH

[M-H] m/z = 426.0
NH2N

AMP

N

ammonium thiosulfate[M-H]- m/z = 113.0[M-H]- m/z=81.0

In addition to carboxylic
acids, the Intrada Organic
Acid column is also able toanalyze stronger acids, like
p-toluenesulfonic acidsulfuric or sulfonic acids
using IEX mode and MS
SO3Hbenzenesulfonic acid detection.
SO3HNNOOCH2OP OHOH

Inorganic phosporic acid,SO32sodium sulfite which is typically analyzed[M-H] m/z = 346.0(100µg//mL in 5mM tris(2-carboxyethyl)phosphine), 0.1%HCOOH)
using ion-chromatography,
345678910 min
12345 min 678
can also be easily separated 05µL (100µg/mL in 0.1% HCOOH)
5µL (50-100µg/mL in 0.1% HCOOH)
A09 on this column.

910A03

Analysis of sugar acids using General Conditions (IEX mode) and high temperatures
50°C
Sugar acid

CH2OHCHOH

HOOCOHOHO CHOHNHCOCH2OH

HOOCOH
OH

CH2OH
CHOH
O CHOHNHCOCH3CH2OH
OHOH
COOH
HOOHOH

01

N-acetylneuraminic acid
[M-H]- m/z = 308.1

2-keto-D-gluconic acid(hemicalcium salt)

60°C
Uronic acid isomers
HOCOOHOOH

galacturonic acid

Sugar acids show poor
peak shape under normal
temperaturesduetoanomerization
ofthealdehyde structure typicallyfound on reducing sugars.

Better peak shapes can beachieved using IEX underelevatedtemperatures
CH2OH
around 50-60°C.
2-keto-3-deoxyoctulosonicacid
O CHOHAn additional benefit is that(ammonium salt)
under high temperatureOH[M-H]- m/z = 237.0 conditions,isomers likeglucuronic and galacturonic
2345 min 6789
10acid epimers may also be
5µL (100µg/mL in 0.1% HCOOH)
A10 separated.
O

HOOC

Sugar acids can also be
analyzed on the Intrada
Organic Acid column, using
N-glycolylneuraminic acidIEX
mode
andhigh[M-H]- m/z = 324.1 temperatures.

OHOH

glucuronic acid
COOHOOHOHHOOH[M-H]- m/z = 193.0

3[M-H] m/z=193.00

01234565µL (40-60µg/mL in 0.1% HCOOH)

7 min
A08

Importance of sample and solvent preparation
When using Intrada Organic Acid, special care should be given to preparation of the sample solvent, especially pH.
This is particularly important when analyzing compounds with low pKa values or polyanionic species, wherepreparation of sample solvents with different pHs can lead to inconsistent results. Proper preparation of the sample
itself is also important, to increase column performance and lifetime, paying particular attention to the removal oflarge polymers such as proteins.

Sample prep using ultrafiltration

Effect of variations in sample solvent pH

Sample preparation for organic acids isusually quite difficult. Here we show a simple
Multiple dicarboxylic acids can vary fast method to prepare samples for injection
dramatically from each other in pKa onto the Intrada Organic Acid column. Thevalues, such as oxalic acid (pKa 1.2, example below shows protein removal using
4.2) or methylmalonic acid (pKa 3.1, centrifugal ultrafiltration (exclusion MW 3kDa).
5.7), which may lead to poor peak Samples may also be treated with 0.2N
shape in formic acid, due to the HClO4 for protein precipitation followed bydifficulty in controlling dissociation centrifugation (13krpm, 10min). The proteinevents. In this case, raising or free supernatant can then be safely injected
lowering sample solvent pH may be onto your column.
used to improve peak shape.
Japanese Sake
Homogeneous dissociation states
20-fold dilution with 0.1% HCOOH
should be targeted with the use ofdifferent solvents.
Centrifugal Ultrafiltration

General Conditions (IEX mode)
5µL (500µg/mL)[M-H] m/z = 89.0

HOOC COOH

oxalic acid
0.02% NH4OH

50mM HCOONH41% HCOOH(Amicon, 3000 NMWL)

Organic acids in Sake

Filtrate (Injection Sample)

General Conditions (IEX mode)
fumaric acid

0.2N HClO4012

3456789

lactic acid

10 min[M-H]- m/z = 115.0

citric acid
[M-H]- m/z = 191.0

A06

5µL (100µg/mL)[M-H] m/z = 117.0

malic acid

CH3
HOOC CH COOH

[M-H] m/z = 117.0

methylmalonic acidphosphoric acid
[M-H]- m/z = 133.0[M-H] m/z = 97.0pyroglutamic acidsuccinic acid

[M-H] m/z = 128.0[M-H] m/z = 89.0

pyruvic acid
01234567[M-H]- m/z = 87.0

8910 min

A11A04

Additional Considerations-The Intrada Organic Acid column should only be used with LC-MS. This product is not recommended for UV, Electrical
Conductivity Detector or ELSD detection, in order to avoid peak identification failure.
While this product is ideal for most organic acids, we cannot guarantee that it will work for all of them, particularly for someisomers.
Detection sensitivity is highly dependent on the structure of the compound as well as the capabilities and setup of the MS
instrument, especially the drying gas temperature.
The sample matrix can have a big impact on peak shape, so sample preparation and sample solvent should be optimized toimprove peak shape.
The guard cartridges for this product are not metal-free, so we do not recommend using them for phosphoric, chelating orion-suppressing compounds.

PRODUCT INFORMATIONPRODUCT NAME

COLUMN DIMENSIONGUARD SYSTEM

30x2mm, 50x2mm,75x2mm, 100x2mm,150x2mm, 250x2mm

Guard Holder / Cartridge Column
The guard cartridges for this product are not metalfree, so we do not recommend using them forphosphoric, chelating or ion-suppressing compounds.

Spherical pure 3µm silica particles / Specialized stationary phase for organic acid (IEX mode, NP mode) / 300bar max. / Metal-free analysis column

Imtakt USA (info@imtaktusa.com)
North America

Imtakt Corp./JAPAN (info@imtakt.com)
Other Countries