TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS ©2013 Waters Corporation

STREAMLINING ANALYSIS OF DESOGESTREL AND ETHINYL ESTRADIOL ORAL CONTRACEPTIVES WITH UPLC

Margaret Maziarz, Sean M. McCarthy, Michael D. Jones, and Warren B. Potts III

Waters Corporation, 34 Maple Street, Milford, MA USA

INTRODUCTION

The majority of the methods described in literature

for the analysis of Desogestrel and Ethinyl Estradiol

oral contraceptives utilize HPLC instrumentation. The HPLC methods are typically long, consume a high

volume of solvents, and in some cases multiple methods are needed to analyze all the related

substances. Converting and consolidating these HPLC methods to a single UPLC method can reduce analysis

time and mobile phase consumption.

Presented here are our results for consolidating two HPLC methods and one GC method for the related

substances analysis of Desogestrel and Ethinyl

Estradiol to one UPLC method. The UPLC method development strategy used to consolidate the

methods will be discussed. The performance of the UPLC method was measured by evaluating system

suitability results against the system suitability recommendations specified in the USP General

Chapter, <621> Chromatography1. The UPLC method was then used for the analysis of commercially

available Desogestrel and Ethinyl Estradiol tablets to demonstrate applicability of the method in release

testing.

The resulting UPLC method provides 88% reduction in

run time over the three legacy methods, while maintaining the specification for resolution of ≥1.5.

Solvent consumption was reduced by 96% with UPLC. In summary, the UPLC method streamlines laboratory

processes, improving throughput and productivity of pharmaceutical manufacturing facilities.

METHODS

EXPERIMENTAL

Resolution Mixture Solution

Separate Stock solutions prepared in methanol at 0.3

mg/mL included the following API’s and their related

substances:

Stock resolution mixture prepared by transferring an equal

volume of each stock solution to one vial was diluted with

water to 0.016 mg/mL of each constituent.

Tablet Sample Solutions

Tablets containing 0.15 mg of Desogestrel and 0.03 mg of

Ethinyl Estradiol were used in this study. Sample solutions

were prepared by dissolving 25 tablets in 6 mL of methanol

by sonication for 20 minutes. Solutions were then diluted

with 4 mL of water and sonicated for an additional 10

minutes.

Legacy HPLC Methods

Parameter HPLC Method 1 HPLC Method 2

LC SystemAlliance® 2695 HPLC with 2489 UV/Visible detector

Alliance® 2695 HPLC with 2489 UV/Visible detector

ColumnSynergi C18 Hydro-RP4.6 x 250 mm, 4 µm

Spherisorb ODS-24.6 x 250 mm, 3 µm

Column Temp. 25°C 25°C

Flow rate 2.0 mL/min 1.5 mL/min

Injection Vol. 25.0 µL 20.0 µL

Mobile Phase

Solvent A42:58 acetonitrile:water

Solvent B75:25 acetontrile:water

Solvent A25:25:50 acetonitrile:methanol:water

Solvent B50:50 acetontrile:methanol

Separation Gradient Gradient

Run Time 48.1 minutes 50.0 minutes

Analysis of (Peak ID)

1, 2, 3, 4, 8, 9 5, 6

Table 1. Summary of the legacy HPLC methods for related

substances analysis of Desogestrel and Ethinyl Estradiol. The GC method was developed for the detection of Desogestrel

Impurity A, however it was not tested in this study.

HPLC Methods

We began our study by reproducing the legacy HPLC methods

on the Alliance HPLC system as shown in Figure 1.

The performance of the method was measured by evaluating

system suitability of the five replicate injections of the resolution

mixture solution against the recommendation specified in the

USP General Chapter, <621> Chromatography. The UPLC system

suitability results for each component can be found in Table 3.

The retention times and area repeatability were well below the

USP specification of 2% RSD for data from five replicate

injections. The resolution between all of the peaks was ≥1.5.

Sample Solutions Analysis

The UPLC method was applied for analysis of the drug tablet formulation. The UPLC data acquired using UV at 210 nm is displayed in

Figure 3. Due to the sensitivity challenge with UV, we explored FLR detector as shown in Figure 4. Two related substances, 5 and 6,

were detected by FLR detector.

Figure 3. UPLC method with UV at 210 nm. Sample solutions

analysis performed on the ACQUITY UPLC H-Class system using

an ACQUITY UPLC HSS T3, 2.1 x 150 mm, 1.8 µm column.

AU

0.000

0.015

0.030

0.045

0.060

Minutes

0.00 1.50 3.00 4.50 6.00 7.50 9.00 10.50 12.00 13.50

7.700 10.439

11.285

11.908

AU

0.000

0.015

0.030

0.045

0.060

Minutes

0.00 1.50 3.00 4.50 6.00 7.50 9.00 10.50 12.00 13.50

3.082

4.621

4.883

5.866

6.265

6.992

7.697

7.941

10.464

11.298

11.802

AU

0.00

0.60

1.20

1.80

2.40

Minutes

0.00 1.50 3.00 4.50 6.00 7.50 9.00 10.50 12.00 13.50

-1

-2

-E

E

-8

-9

DE

S

UPLC Data with UV at 210 nm

Diluent blank

Drug tablet solution

Placebo tablet solution

AU

0.00

0.05

0.10

0.15

Minutes0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00

1

23

EE

4

8

9

DES

AU

0.00

0.08

0.16

0.24

Minutes0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00

5 6

EE

a. HPLC method 1

b. HPLC method 2

Figure 1. Legacy HPLC methods performed on the Alliance 2695

system. UV at 210 nm. (a) HPLC method 1 for analysis of 6 re-

lated substances of Ethinyl Estradiol and Desogestrel and (b)

HPLC method 2 for the detection of an additional two related

substances. The HPLC methods provided acceptable resolution

between components; however the total analysis for these two

methods was 98 minutes. This time increased when factoring in

the need for an additional GC analysis, which is not shown.

Table 4. UPLC results with UV at 210nm. Drug tablet sample

solutions analysis performed on the ACQUITY UPLC H-Class

system using an ACQUITY UPLC HSS T3, 2.1 x 150 mm, 1.8

µm column. The reported %Area reflects the peak area of the

related substance over the peak area of the corresponding API.

Table 5. UPLC results with FLR. Drug tablet sample solution

analysis performed on the ACQUITY UPLC H-Class system using

an ACQUITY UPLC HSS T3, 2.1 x 150 mm, 1.8 µm column. The

reported %Area reflects the peak area of the related substance

over the peak area of the corresponding API.

Method Development

The legacy methods were consolidated to one UPLC method.

Due to the complexity of combining three analytical approaches,

we followed a method development approach to resolve

constituents with a goal of achieving a resolution ≥ 1.5

between all the known peaks. The UPLC method development

was conducted using water (solvent A) and acetonitrile (solvent

B) due to the neutral characteristics of the analytes. Different

stationary phases were explored such as ACQUITY UPLC BEH,

HSS T3, BEH Phenyl, and BEH Shield. The ACQUITY UPLC HSS

T3 column provided the best separation. Temperatures, various

gradient elution conditions, and flow rates were also explored to

further optimize separation between all the peaks.

Figure 4. UPLC method with FLR. Sample solutions analysis per-

formed on the ACQUITY UPLC H-Class system using an ACQUITY

UPLC HSS T3, 2.1 x 150 mm, 1.8 µm column.

UPLC Data with FLR

EU

0.00

1.50

3.00

4.50

Minutes

0.00 1.50 3.00 4.50 6.00 7.50 9.00 10.50 12.00 13.50

EU

0.00

1.50

3.00

4.50

Minutes

0.00 1.50 3.00 4.50 6.00 7.50 9.00 10.50 12.00 13.50

EU

0.00

1.50

3.00

4.50

Minutes

0.00 1.50 3.00 4.50 6.00 7.50 9.00 10.50 12.00 13.50

4.9

00 -

EE

2.3

97 -

62.2

02 -

5

Drug tablet solution

Placebo tablet solution

Diluent blank

CONCLUSION Three legacy methods, two HPLC and one GC, for related substances analysis of Desogestrel and Ethinyl Estradiol were successfully consolidated to one UPLC method

The UPLC method met the system suitability recommendations defined in the USP General Chapter, <621> Chromatography

― Repeatability of the retention times and peak areas were below the USP specification of 2% RSD and resolution between all of the peaks was ≥1.5

The resulted UPLC method

― Decreased run time by 88% over the three legacy methods, reduced solvent consumption by 96%, and eliminated the need for a GC instrumentation

RESULTS

Table 3. System Suitability results for 5 replicate injections of

the resolution mixture solution acquired on the ACQUITY UPLC H-Class system using an ACQUITY UPLC HSS T3, 2.1 x 150

mm, 1.8 µm column.

References

1. USP General Chapter, <621> Chromatography, USP35-NF30, The United States Pharmacopeia Convention, official December 1, 2012

Parameter UPLC Method

LC SystemACQUITY UPLC® H-Class with PDA and FLR detectors

ColumnACQUITY UPLC HSS T32.1 x 150 mm, 1.8 µm

Column Temp. 67°C

Flow rate 0.4 mL/min

Injection Vol. 5.0 µL

Detection UV, 210 nmFLR, λex: 208 nm, λem: 210 nm

Run Time 18.6 min

Mobile Phase Solvent A: 100% water

Solvent B: 100% acetontrile

Separation Gradient

Wash Solvents 50:50 water:acetonitrile

Sample Temp. 20°C

StepTime

(minutes)%A %B Curve

1 Initial 67.0 33.0 Initial

2 10.0 14.0 86.0 6

3 13.5 14.0 86.0 6

4 13.6 67.0 33.0 6

5 18.6 67.0 33.0 6

Table 2. UPLC method conditions for related substances

analysis of Desogestrel and Ethinyl Estradiol.

UPLC Method

AU

0.00

0.05

0.10

0.15

0.20

Minutes

0.00 1.50 3.00 4.50 6.00 7.50 9.00 10.50 12.00 13.50

5 6 1

2

EE 3 4

8

9

7

DES

UPLC Method

Figure 2. UPLC method with UV at 210 nm. Resolution mixture so-

lution of API’s and related substances performed on the ACQUITY UPLC H-Class system using an ACQUITY UPLC HSS T3, 2.1 x 150

mm, 1.8 µm column.

Analytes Peak ID

Ethinyl Estradiol (API) EE

6-keto-Ethinyl Estradiol 1

Δ 9,11-Ethinyl Estradiol 2

Estrone 3

17 β-Ethinyl Estradiol 4

6 α-Ethinyl Estradiol 5

6 β-Ethinyl Estradiol 6

Desogestrel (API) DES

Desogestrel Impurity A 7

Desogestrel Impurity B 8

Desogestrel Impurity C 9