Method Optimization of Hydroxychloroquine
in Reversed Phase Mode- Rapid Analysis Using Volatile Mobile Phase -（F140925JE）

Introduction
Hydroxychloroquine was first approved in the United States in 1955. And nowadays, it has
been approved in more than 70 countries all over the world as a remedy of malaria,malaria systemiclupus erythematosus, cutaneous lupus erythematosus and a variety of disease.

Official HPLC analytical method of Hydroxychloroquine is stipulated in the USP (The UnitedStates Pharmacopeia), section of “Hydroxychloroquine Sulfate Tablets, part ofAssay”.

However the USP method cannot be used with LC
However,
LC-MS(/MS)
MS(/MS) because a nonnon-volatile
volatile ionion-pairing
pairing
reagent is used.

In this technical data sheet, we will introduce method optimization for rapid analysis of
Hydroxychloroquine and Chloroquine mixture using organic/inorganic hybrid silica column,
Triart C18,
C18 and volatile mobile phase that is compatible with LC
LC-MS(/MS)
MS(/MS).

2

Mobile Phase Screening with Triart C18
HPLC Conditions
Column： YMC-Triart C18 1.9 µm

Eluent：(50 X 2.0 mmI.D)

A) 20 mM formate bufferB) acetonitrile or methanol
5-95%B (0-10 min), 5%B (10-20 min)：Flow rate

Temperat re ：Temperature

0.2 mL/min
25℃Detection：

UV at 254 nm

Injection：

4 µL (50 µg/mL)

Sample：1 Hydroxychloroquine sulfate
1.
2. Chloroquine phosphate

NHOHNN

CH 3

CH 3

Hydroxychloroquine sulfate(pKa1 = 7.28)(pKa2 = 9.76)

・H2SO4ClNHN
N

CH3

CH 3
CH 3

・H3PO4Chloroquine phosphate(pKa1 = 7.29)(pKa2 = 10.32)

Cl3

Mobile Phase Screening
Triart C18 X pH X Organic Solventacetonitrile（Gradient）
mAU

pH 2.96020 mM
HCOOH40methanol（Gradient）

160

20 ¥140416_TA_00010.D
220 mMHCOOH-HCOONH440

46810min*

12mAU

pH 4.320 mM
HCOONH410min*

6810min*

810min*120 ¥140416_TA_00104.D
246810min*

0mAU

6060

1240

244020Peak shape was deteriorated
atpH6.1 and pH9.9 because
they are0810min*
02near pKa values of the compounds.

2012¥140415_TA_00010.D

24646mAU

60

160

140402220200 ¥140415_TA_00022.D0820mAU20 mM
HCOONH4-NH36Select pH 4.30 ¥140415_TA_00004.DpH 9.9440mAU02600 ¥140416_TA_00110.DpH 6.10mAU

2002200 ¥140416_TA_00004.D60

1402001. Hydroxychloroquine sulfate
2. Chloroquine phosphate

mAU0 ¥140415_TA_00016.D
246810min*0246810min*4

Mobile Phase Optimization
Selection of Organic Solvent and Adjustment of Compositionacetonitrile（Gradient）

Result of screening

methanol（Gradient）
1. Hydroxychloroquine sulfate
2. Chloroquine phosphate

pH 4.320 mM
HCOOH-HCOONH4CH3CN(%)

12mAU60

4040202550 ¥140416_TA_00110.D
0246810min*

1mAU

9560

MeOH
(%)
952362050 ¥140416_TA_00104.D
0246810min*

Elution method change from Gradient to Isocratic
Tips

It is effective to choose 15 to 20% lower organic composition thanwhere a sample is eluted on gradient elution.
e.g. 25% on gradient >>> 25-15=10% on Isocratic

20 mM HCOOH-HCOONH4 (pH 4.3)/acetonitrile (90/10)

Rs (1,2) = 5.7

1mAU

Compared to methanol

20・shorter analysis time・better resolution

21020 mM HCOOH-HCOONH4 (pH 4.3)/methanol (85/15)

Rs (1,2) = 4.4mAU
20

1102

Proceed to speeding up
000F141009C32468101214 min

0F141009D

2468101214 min

5Speeding Up of Analysis
Column : Triart C18 1.9 µm, 50 X 2.0 mmI.D.
Eluent : 20 mM HCOOH-HCOONH4 (pH 4.3)/acetonitrile (90/10)
0.2 mL/min

1. Hydroxychloroquine sulfate
2. Chloroquine phosphate

16.6 MPa(2,410 psi)
F141009C

8.0 min

0.4 mL/min

Temperature : 25℃Detection: UV at 254 nmInjection: 2 µL (10 µg/mL)

31.0 MPa(4,500 psi)
4.0 min

F140724E

0.6 mL/min
44.3 MPa(6,430 psi)
1
2.6 min

F140724F

0.8 mL/min

257.2
572 MPa(8,300 psi)
2.0 min

F140724G

1.0 mL/min

0.8

1.0

1.2

1.4

69.0 MPa(10,000 psi)

1.6 min

F140724H01234567min

6Comparison with USP Method
YMC method：volatile mobile phase, UHPLC column
Triart C18 1.9 µm, 50 X 2.0 mmI.D.

12NCH 3・H2SO4Hydroxychloroquine sulfate

LC-MS(MS) compatible

102011.01.2

1.4

1.6 minmin: acetonitrile/methanol/water/phosphoric acid(100/100/800/2)
containing 96 mg of sodium 1-pentanesulfonate
Flow rate: 1.0 mL/min
Temperature : 25℃
Detection: UV at 254 nm
Injection: 20 µL (50 µg/mL)
Pressure: 9.5 MPa (1,380 psi)

1150

2

100

50F140514F

246

Chloroquine phosphate

EluentmAU0・H3PO4: 20 mM HCOOH-HCOONH4 (pH 4.3)/acetonitrile(90/10)
Flow rate: 1.0 mL/min
Temperature : 25℃
D t tiDetection: UV att 254 nmInjection: 2 µL (10 µg/mL)
Pressure: 69.0 MPa (10,000 psi)

USP method：non-volatile mobile phase, conventional column
Triart C18 5 µm, 250 X 4.6 mmI.D.

0CH 3Eluent

50.8

ClCH 3Analysis time: 1/10 of USP method

mAU

F140724H

CH3CH 3Cl0NNN1NHOHNH81012141618min7