The Japanese Pharmacopoeia,
18th Edition Analysis of tranexamic acidLC Technical Note

LT195

－ 2.00 Change of conditions specified within General theory of chromatography－GL Sciences Inc.

Adjustable range of chromatography conditions has been specified in [2.00 General theory of chromatography]
which has been stipulated based on the contents harmonized and agreed upon by Japanese/U.S./European
Trilateral Pharmacopoeia at the first supplemental public comment of the Japanese Pharmacopoeia,
18th Edition, which was disclosed in September 2021.
Based on this, the speed-up of analysis can be expected by optimizing the column size and analytical conditions
within the acceptable changeable range.
In this report, an example of analysis was reported, in which analysis time is shortened and solvents consumption
is reduced by changing the conditions within the range specified in [2.00 General theory of chromatography]
for JP18 Test for Quantitative Method of Tranexamic Acid.
* The change of analytical conditions is for the content to be applied from the first supplemental revision of JP18 andis not for the content announced in the past. Accordingly, this Technical Note is simply just for reference.

Changeable items in JP
In [2.00 General theory of chromatography], the change of LC column and instrumental
conditions is allowed by satisfying the requirements of system suitability.
Japanese Pharmacopoeia<Public comment proposal in September 2021>
International harmonization of Trilateral pharmacopoeia
ColumnInstrument

Mobilephase

Functionalgroup
Particlediameter
LengthNon-changeable
Totally porous particle: Specified ratio of L/dp is changeable within the range of- 25% - +50%
Surface porous particle: Combination of other L and dp is allowed if theoretical plate
number is within the range of - 25% - +50% of specified column.

Inner diameter

If there is no change in particle diameter or column length, an adjustment to thecolumn inner diameter may be needed.

Flow rate

By changing both column inner diameter and particle diameter, changeable by theequation below.
F2 = F1 × [(dc22 × dp1) / (dc12 × dp2)]* There are detailed provisions if the diameter crosses 3 um and after adjustment bychanging the column particle diameter and the column length, the change is allowed
within the range of +/-50%.

Injection volume

When changing the column particle diameter and the length, injection volume can beadjusted by the equation below.
Vinj2 = Vinj1 × (L2 × dc22) / (L1 × dc12)

Detection
wavelength

Non-changeable

Columntemperature+/-10℃

pH+/-0.2

Buffer solution
temperature+/-10% within changeable pH range

Mixing ratio+/-30%(relative) for mobile phase with the composition of less than 50%* Within +/-10% against total

GL Sciences LC Technical Note

Flow rate

Injection volume

F1 ：Flow rate of each medicinal product (mL/min)
Vinj1 ：Injection volume of each medicinal product (μL)
F2 ：Flow rate after change (mL/min)
Vinj2 ：Adjusted injection volume (μL)
dp1 ：Column particle diameter of each medicinal product (mm) L1 ：Column length of each medicinal product (cm)dp2 ：Particle diameter of column to use (mm)
L2 ：New column length (cm)dc1 ：Column inner diameter of each medicinal product (mm)* dc is synonymous with flow rate
dc2 ：Column diameter to be used (mm)GL Sciences LC Technical Note
Example: JP18 Tranexamic Acid Quantitation MethodReferring to JP18 Tranexamic Acid Test as an analysis example, the column size was changed
within the changeable range specified in [2.00 General theory of chromatography] and theanalysis was implemented trying to find the speed-up conditions for analysis.
<Test conditions for JP18 Tranexamic Acid Quantitative Method>
Detector: UV absorption photometer (measuring wavelength: 220nm)
Column: Octadecylsilylated silica gel with 5 μm particle size for liquid chromatography is packed in astainless steel tube with 6.0mm inner diameter and 25cm length.
Column temperature: Constant temperature at around 25℃
Mobile phase: 11.0g of anhydrous sodium dihydrogen phosphate is dissolvedin 500ml water, and 5mL of triethylamine and 1.4g of sodium lauryl sulphate are added.
After adjusting pH of the solution to pH 2.5 by using phosphoric acid or phosphoricacid solution (1 10), water is added to make the volume to be 600mL. Then, 400mL of methanol isadded.
Flow rate: Flow rate is adjusted so that the retention time of tranexamic acid may be about 20 min.
* Using InertSustain AQ-C18, the result was compared with the application in which
the retention time was adjusted by 1.4mL/min.
Injection volume: 20 μL* For details, refer to our Technical Note LT092.
https://www.gls.co.jp/technique/app/detail.php?data_number=LT092

The column size written in JP is 25cm in column length (L) and 5μm in particle diameter (dp),and therefore, the value of L/dp becomes 50,000.
The changeable range of L/dp is within -25% - +50%, and so the changeable range of L/dp
becomes as follows.

37,500 ≦ L/dp ≦ 75,000
This time, the column of 3μm in particle diameter (dp) and 3.0mm in inner diameter (dc)
was selected to set up the speed-up conditions as in the table below.

JPconditions

Changeable range

Speed-upconditions

Column length (L, mm)

250 ㎜

113 – 225mm

150 mmColumn particle diameter(dp, μm)

5 μm

-

3 μm

L/dp

50,00037,500 – 75,000

50,000Inner diameter (dc, mm)

6.0 mm-

3.0 mmFlow rate (F, mL/min)

1.4 mL/min

0.35 – 1.06mL/min

0.35mL/min

Injection volume (Vinj, μL)

20 μL

3 μL

3 μL

GL Sciences LC Technical Note
Conditions written in JP <before change>
1. Tranexamic acid (200 mg/L)
2. 4-Aminobenzoic acid methyl ester (2 mg/L)

HPLC conditions

mAU
2: InertSustain AQ-C18(5 μm, 250 x 6.0 mmI.D.)
EluentA) Phosphoric acid buffer solution*1
B) CH3OH
A/B = 60/40, v/v
Temperature: 25 ℃
Detector: UV 220 nm
Injection volume : 20 μL
Flow rate: 1.4 mL/min

4Column

10

RT : 20.4 min→0Analysis time ： About 30 min.
Volume of solvent used： About 42mL

210Time (min)2030

Speed-up conditions <after change>
HPLC conditions
Column21
RT : 11.1 min→0mAU
2

4: InertSustain AQ-C18 HP: (3 μm, 150 x 3.0 mmI.D.)
EluentA) Phosphoric acid buffer solution*1B) CH3OH: A/B = 60/40, v/v
Temperature: 25 ℃Detector: UV 220 nm
Injection volume : 3 μL
Flow rate: 0.35 mL/min

1. Tranexamic acid (200 mg/L)
2. 4-Aminobenzoic acid methyl ester (2 mg/L)

0Analysis time ： About 15 min.
Volume of solvent used： About 5.25mL[Analysis time, shortened toapprox. 1/2] and [Volume ofsolvent used, saved to approx. 1/8]
were achieved.
By downsizing the column, it isexpected that equilibration
time of mobile phase can beshortened.
*1 Phosphoric acid buffer solution
Refer to the test conditions in the previous page.

10Time (min)2030<Result of system suitability test>
Criteria

Conditions
written in JP

Speed-upconditions

Resolution(1,2)

More than
5

6.4

PASS

5.3

PASS

RSD%(1)(n=6)

Less than
0.6%

0.02%

PASS

0.55%

PASS

The good results for the conditions written in JP and thespeed-up conditions were obtained as shown in thetable above, by satisfying the requirements specified in
the system suitability for both conditions.

GL Sciences LC Technical NoteStructure
COOHCOOH

H2N

H2N

Tranexamic acid

4-aminomethylbenzoic acid
Structures are created using Chemistry 4-D Draw which is provided by ChemInnovation Software, Inc.

*This data is for reference only when selecting the columns for the customer
considering the analysis based on pharmacopoeia and is not to guarantee the system
suitability of the customer’s instruments.

Products used
●Analytical column
InertSustain AQ-C18 HP
3 μm, 150 x 3.0 mm I.D.
Cat.No. 5020-89930

GL Sciences disclaims any and all responsibility for any injury or damage which may be caused by this data directly or indirectly.
We reserve the right to amend this information or data at any time and without any prior announcement.

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