TN-1275

APPLICATIONS Zeshan Aqeel

Senior Application Scientist
Zeshan loves to collect watches
European Pharmacopeia Monograph 2287 and the Back to the Future Trilogy.
He has twin boys and a baby
Fluconazole: Efficient Separation on Fully Porous girl which drive him crazy! He is
an Apple Fanboy for life and he

and Core-Shell C18 Columns Within the Allowable likes being in the lab more than
anywhere else.

Adjustments
Zeshan Aqeel1, Dirk Hansen2, and Heiko Behr2
1
Phenomenex, Inc., 411 Madrid Avenue, Torrance, CA 90501 USA
2
Phenomenex Ltd. Deutschland, Zeppelinstraße 5, 63741 Aschaffenburg, Germany

Introduction Conditions
Fluconazole [2-(2,4-Difluorophenyl)-1,3-bis(1H-1,2,4-triazol-1-yl) LC-UV Conditions
propan-2-ol] is a triazole based systemic antifungal medication. Luna® 5 µm C18(2) 150 x 4.6 mm (00F-4252-E0)
Columns:
It belongs to the first-generation antifungals used for oral treat- Kinetex® 2.6 µm C18 100 x 4.6 mm (00D-4462-E0)
ment of Candida infections. The desirable pharmacologic prop- Isocratic (84:16)
Mobile Phase:
erties of Fluconazole include a relatively long half-life, and the A: 0.63 g/L Ammonium formate in water
ability to be administered orally or parentally1. The monograph B: Acetonitrile
for related substances of Fluconazole specifies three main impu- Flow Rate: 1.0 mL/min
rities (A [2(RS)-2-(2,4-Difluorophenyl)-1-(1H-1,2,4-triazol-1-yl)-3- Temperature: 40 °C
(4H-1,2,4-triazol-4yl)propan-2-ol], B {2-[2-fluoro-4-(1H-1,2,4-tri-
Detection: UV @ 260 nm
azol-1-yl)phenyl]-1,3-bis(1H-1,2,4-triazol-1yl)propan-2-ol}, and
Injection Volume: 20 µL
C [1,1’-(1,3-phenylene)di-1H-1,2,4-triazole]) which need to be
separated and detected to demonstrate compliance2. HPLC System: Agilent® 1260 (Agilent Technologies®, Santa Clara, CA,
USA)

Table 1
Preparation of Test and Reference Solutions
Solution Step 1 Step 2 Step 3 Final Conc.
Test Solution 100 mg Fluconazole dissolve in mobile phase sonicate if necessary and dilute to 10 mg/mL Fluconazole
10.0 mL with mobile phase
Ref a dilute 5.0 mL Test Solution with take 1.0 mL dilute with mobile phase to 10.0 mL 0.05 mg/mL Fluconazole
mobile phase to 100.0 mL
Ref b 5 mg Fluconazole for Peak dissolve in mobile phase sonicate if necessary and dilute to 0.5 mg/mL Fluconazole
Identification 10.0 mL with mobile phase
Ref c 3.0 mg Fluconazole impurity B dissolve in mobile phase sonicate if necessary and dilute with 0.03 mg/mL impurity B
mobile phase to 100.0 mL
Ref d 3.0 mg Fluconazole impurity C dissolve in mobile phase sonicate if necessary and dilute with 0.15 mg/mL impurity C
mobile phase to 20.0 mL
Ref e 1.0 mL Ref d add 1.0 mL Test Solution dilute with mobile phase to 10.0 mL 0.015 mg/mL impurity C
1 mg/mL Fluconazole
Ref f 1.0 mL Ref d dilute with mobile phase to 10.0 mL 0.015 mg/mL impurity C

Results
The test solution and reference solutions (a) to (f) have been 2.71 min on Kinetex 2.6 µm C18. Impurity B is identified through
prepared as indicated in the monograph for Fluconazole2 and injection of reference solution (c) shown in Figures 4a and 4b.
detailed in Table 1. Figures 2a and 2b show the chromatograms The retention time for this impurity is 5.58 min on Luna 5 µm
for the peak identification of the Fluconazole peak. The respec- C18(2) and 2.49 min on Kinetex 2.6 µm C18. The last impurity –
tive retention times achieved are 12.86 min on Luna 5 µm C18(2) impurity C – is identified through injection of reference solution (f)
and 5.12 min on Kinetex 2.6 µm C18. The peak identification of shown in Figures 7a and 7b. The respective retention times for
impurity A and Fluconazole is shown in Figures 3a and 3b. The impurity C are 10.69 min on Luna 5 µm C18(2) and 8.34 min on
retention time for impurity A is 6.33 min on Luna 5 µm C18(2) and Kinetex 2.6 µm C18.

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TN-1275

APPLICATIONS
Test and Reference solutions
Test solution
100 mg of Fluconazole was dissolved in mobile phase, sonicated and diluted to 10 mL with mobile phase – Injection: 20 µL

Figure 1a Figure 1b
Test Solution (10 mg/mL of Fluconazole CRS) on Luna® 5 µm C18(2) Test Solution (10 mg/mL of Fluconazole CRS) on Kinetex® 2.6 µm C18
150 x 4.6 mm 100 x 4.6 mm

mAU mAU
3 3
1

25 25

20
20

15
15

App ID 26029
App ID 26022
1 10
10

5 2
5

2
0
0

0 2 4 6 8 10 12 14 16 min 0 2 4 6 8 10 12 14 16 min

Peak Peak
No. Analyte Time Area Height Width Area% Symmetry No. Analyte Time Area Height Width Area% Symmetry
1 Impurity B 5.579 74.2 9.3 0.1209 0.525 0.935 1 Impurity B 2.489 73.8 25.7 0.0456 0.521 0.814

2 Impurity A 6.33 11 1.2 0.1408 0.078 0.841 2 Impurity A 2.703 11.3 3.4 0.0506 0.080 0.868

3 Fluconazole 12.256 14052.7 506.8 0.3875 99.397 0.214 3 Fluconazole 4.744 14075.9 912.3 0.208 99.399 0.146

Reference solution (a)

5 mL of the test solution was diluted to 100 mL with the mobile phase. 1 mL of this solution was then diluted to 10 mL with the mobile phase
– Injection: 20 µL.

Figure 2a Figure 2b
Reference solution (a) on Luna 5 µm C18(2) on Agilent® 1260 Reference solution (a) on Kinetex 2.6 µm C18 on Agilent 1260
mAU

mAU
12
1

12
10

10
8

8
App ID 26023

6
App ID 26030

1 6
4
4

2
2

0
0

0 2 4 6 8 10 12 14 16 min 0 2 4 6 8 10 12 14 16 min

Peak Peak
No. Analyte Time Area Height Width Area% Symmetry No. Analyte Time Area Height Width Area% Symmetry
1 Fluconazole 12.86 77.4 4.3 0.2719 100.000 0.93 1 Fluconazole 5.12 72.8 12.6 0.0915 100.000 0.812

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Reference solution (b)

5 mg of Fluconazole for peak identification CRS (containing impurity A) was dissolved in mobile phase, sonicated and diluted to 10 mL with
the mobile phase—Injection: 20 µL.

Figure 3a Figure 3b
Reference solution (b) on Luna® 5 µm C18(2) on Agilent® 1260 Reference solution (b) on Kinetex® 2.6 µm C18 on Agilent 1260

mAU 2 mAU 2

3 3

2 2

App ID 26024

App ID 26031
1 1 1

0 0

0 2 4 6 8 10 12 14 16 min 0 2 4 6 8 10 12 14 16 min

Peak Peak
No. Analyte Time Area Height Width Area% Symmetry No. Analyte Time Area Height Width Area% Symmetry
1 Impurity A 6.332 7.4 8.3E-1 0.1477 1.307 0.987 1 Impurity A 2.709 6.5 2.1 0.052 1.149 1.191

2 Fluconazole 12.826 558.4 32.7 0.2633 98.693 0.857 2 Fluconazole 5.088 558.5 94.2 0.0892 98.851 0.623

Reference solution (c)

3.0 mg of Fluconazole impurity B CRS was dissolved in the mobile phase, sonicated and then diluted to 100 mL with mobile phase—
Injection: 20 µL.

Figure 4a Figure 4b
Reference solution (c) on Luna 5 µm C18(2) on Agilent 1260 Reference solution (c) on Kinetex 2.6 µm C18 on Agilent 1260
mAU mAU
1

140 140

120 120

100 100

80 80
App ID 26025

App ID 26032

1 60
60

40
40

20
20

0
0

0 2 4 6 8 10 12 14 16
0 2 4 6 8 10 12 14 16 min

Peak Peak
No. Analyte Time Area Height Width Area% Symmetry No. Analyte Time Area Height Width Area% Symmetry
1 Impurity B 5.582 458.8 57.4 0.1213 100.000 0.918 1 Impurity B 2.493 457.7 143.3 0.0533 100.000 0.762

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TN-1275

APPLICATIONS
Reference solution (d)
3.0 mg of Fluconazole impurity C CRS was dissolved in the mobile phase, sonicated and then diluted to 20 mL with mobile phase—
Injection: 20 µL

Figure 5a Figure 5b
Reference solution (d) on Luna® 5 µm C18(2) on Agilent® 1260 Reference solution (d) on Kinetex® 2.6 µm C18 on Agilent 1260
mAU mAU

120 120 1

100 100

80 80

60 60

App ID 26026

App ID 26033
1
40 40

20 20

0 0

0 2 4 6 8 10 12 14 16 min 0 2 4 6 8 10 12 14 16 min

Peak Peak
No. Analyte Time Area Height Width Area% Symmetry No. Analyte Time Area Height Width Area% Symmetry
1 Impurity C 10.684 582.2 41.6 0.2153 100.000 0.935 1 Impurity C 4.418 580.3 115 0.0789 100.000 0.824

Reference solution (e)

System suitability: A mixture of 1 mL of reference solution (d) and 1 mL of test solution were diluted to 10 mL with the solvent mixture.
Injection: 20 µL.
Figure 6a Figure 6b
Reference solution (e) on Luna 5 µm C18(2) on Agilent 1260 Reference solution (e) on Kinetex 2.6 µm C18 on Agilent 1260
mAU mAU
14
14

12
12

10
10

8
8
App ID 26027

6
6

App ID 26034
4
4

2
2

0
0

0 2 4 6 8 10 12 14 16 min
0 2 4 6 8 10 12 14 16 min

Peak Peak
No. Analyte Time Area Height Width Area% Symmetry No. Analyte Time Area Height Width Area% Symmetry
1 Impurity C 10.678 59.1 4.1 0.2227 3.998 0.932 1 Impurity C 4.411 58.6 11.6 0.0791 3.977 0.821

2 Fluconazole 12.769 1418.5 81.4 0.2674 96.002 0.697 2 Fluconazole 5.04 1414.2 210.8 0.0983 96.023 0.409

Table 2 Table 3
Reference solution (e) on Luna 5 µm C18(2) 150 x 4.6 mm Reference solution (e) on Kinetex 2.6 µm C18 100 x 4.6 mm
Inj. # Analyte Time Area Height Width Resolution Inj. # Analyte Time Area Height Width Resolution
1 1 Impurity C 10.678 59.1 4.1 0.2227 1 1 Impurity C 4.411 58.6 11.6 0.0791
2 Fluconazole 12.769 1418.5 81.4 0.2674 5.15 2 Fluconazole 5.04 1414.2 210.8 0.0983 4.22

2 1 Impurity C 10.695 59.7 4.1 0.2245 2 1 Impurity C 4.413 58.7 11.5 0.0754

2 Fluconazole 12.785 1416.7 81.3 0.2714 5.12 2 Fluconazole 5.043 1411.7 204.2 0.1007 4.22

3 1 Impurity C 10.68 58.2 4.1 0.2216 3 1 Impurity C 4.413 56.2 11.6 0.0708

2 Fluconazole 12.776 1415.2 81.2 0.2674 5.15 2 Fluconazole 5.038 1409.3 209 0.0967 4.2

4 1 Impurity C 10.683 58.8 4.1 0.2175 4 1 Impurity C 4.413 56.4 11.7 0.0709

2 Fluconazole 12.774 1413.7 81.3 0.2668 5.13 2 Fluconazole 5.038 1406.2 209.1 0.0985 4.21

5 1 Impurity C 10.695 62.1 4.2 0.23 5 1 Impurity C 4.414 56.6 11.7 0.0736

2 Fluconazole 12.785 1414.9 81.3 0.271 5.11 2 Fluconazole 5.038 1408.5 209.3 0.0955 4.21

Average 5.132 Average 4.212

STD 0.0178885 STD 0.0083666

%RSD 0.3485687 %RSD 0.1986372

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TN-1275

The Luna® 5 µm C18(2) column and the Kinetex® 2.6 µm C18

column were able to provide a resolution factor between Fluco-
nazole and impurity C above the system suitability requirement
of 3.0. The resolution on Luna 5 µm C18(2) was 5.13 (Table 2).
The resolution on Kinetex 2.6 µm C18 was 4.21 (Table 3).

Reference solution (f)

A mixture of 1 mL of reference solution (d) was diluted to 10 mL with the mobile phase. Injection: 20 µL.
Figure 7a Figure 7b
Reference solution (f) on Luna 5 µm C18(2) on Agilent® 1260 Reference solution (f) on Kinetex 2.6 µm C18 on Agilent 1260
mAU mAU

14
14

12 1
12

10
10

8
8
App ID 26028

6 6
1

App ID 26035
4 4

2 2

0 0

0 2 4 6 8 10 12 14 16 min
0 2 4 6 8 10 12 14 16 min

Peak No. Analyte Time Area Height Width Area% Symmetry Analyte Time Area Height Width Area% Symmetry
1 Impurity C 10.687 62.3 4.2 0.224 100.000 0.914 Impurity C 8.338 60.4 5 0.1842 100.000 0.837

Conclusion References
The above experiments show both Luna 5 µm C18(2) and Kine- 1. Wallace, J. E.; Harris, S. C.; Gallegos, J.; Foulds, G.; Chen, T.
tex 2.6 µm C18 are suitable under the conditions outlined in the J. H.; Rinaldi, M. G., Antimicrobial Agents and Chemotherapy,
monograph for Fluconazole2. With the Kinetex 2.6 µm C18 col- 1992, 36(3), 603-606
umn we demonstrated the possibility to reduce the total analysis
time significantly by reducing the retention time for Fluconazole 2. European Pharmacopoeia; Supplement 10 – Monograph
to 5.0 min within the allowable adjustments of compendial meth- 2287
ods3. Therefore, Luna 5 µm C18(2) and Kinetex 2.6 µm C18 are
3. European Pharmacopoeia; Supplement 10 – Chapter 2.2.46
reliable solutions for the analysis of Fluconazole in routine labo-
Chromatographic Separation Techniques
ratories following the Ph. Eur. regulations.

Sources of Standards
Standard Source
Fluconazole CRS EDQM, Y0000557

Fluconazole for Peak Identification CRS EDQM, Y0000558

(contains Impurity A)
Fluconazole impurity B CRS EDQM, Y0000573

Fluconazole impurity C CRS EDQM, Y0000574

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TN-1275

Ordering Information
Luna® 3 μm MidBore™ and Analytical Columns (mm) SecurityGuard™ Cartridges (mm)
Phases 30 x 3.0 50 x 3.0 150 x 3.0 30 x 4.6 50 x 4.6 75 x 4.6 100 x 4.6 150 x 4.6 4 x 2.0* 4 x 3.0*
/10pk /10pk
C18(2) 00A-4251-Y0 00B-4251-Y0 00F-4251-Y0 00A-4251-E0 00B-4251-E0 00C-4251-E0 00D-4251-E0 00F-4251-E0 AJ0-4286 AJ0-4287
for ID: 2.0-3.0 mm 3.2-8.0 mm

Luna® 5 μm MidBore and Analytical Columns (mm) SecurityGuard™ Cartridges (mm)

Phases 30 x 3.0 50 x 3.0 150 x 3.0 250 x 3.0 30 x 4.6 50 x 4.6 75 x 4.6 4 x 2.0* 4 x 3.0*
/10pk /10pk
Luna C18(2) 00A-4252-Y0 00B-4252-Y0 00F-4252-Y0 00G-4252-Y0 00A-4252-E0 00B-4252-E0 00C-4252-E0 AJ0-4286 AJ0-4287
for ID: 2.0-3.0 mm 3.2-8.0 mm

Luna® 5 μm Analytical and Semi-Prep Columns (mm) SecurityGuard™ Cartridges (mm)

Phases 100 x 4.6 150 x 4.6 250 x 4.6 250 x 10 4 x 3.0* 10 x 10**
/10pk /3pk
Luna C18(2) 00D-4252-E0 00F-4252-E0 00G-4252-E0 00G-4252-N0 AJ0-4287 AJ0-7221
for ID: 3.2-8.0 mm 9-16 mm

SecurityGuard™
Kinetex® 2.6 μm Analytical Columns (mm) ULTRA Cartridges‡
Phases 30 x 4.6 50 x 4.6 75 x 4.6 100 x 4.6 150 x 4.6 250 x 4.6 3/pk
C18 00A-4462-E0 00B-4462-E0 00C-4462-E0 00D-4462-E0 00F-4462-E0 00G-4462-E0 AJ0-8768
for 4.6 mm ID

‡ SecurityGuard ULTRA Cartridges require holder, Part No.: AJ0-9000

* SecurityGuard Analytical Cartridges require holder, Part No.: KJ0-4282
**SemiPREP SecurityGuard Cartridges require holder, Part No.: AJ0-9281

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