118 Chiang Mai J. Sci. 2019; 46(1)

Chiang Mai J. Sci. 2019; 46(1) : 118-131http://epg.science.cmu.ac.th/ejournal/Contributed Paper

High Performance Liquid ChromatographyIncorporating to Short Column and Micellar MobilePhase for Determination of Some ContraceptiveDrugsThanatcha Chaida* [a,b], Suchada Udomwatwong [b], Warawut Tiyapongpattana [b] and

Napaporn Youngvises [b]

[a] Center of Scientific Equipment for Advanced Research, Thammasat University, Pathum Thani,

12120, Thailand.

[b] Innovative Green Chemistry Research Unit, Department of Chemistry, Faculty of Science and

Technology, Thammasat University, Pathum Thani, 12120, Thailand.

* Author for correspondence; e-mail: thanatcha.cha@gmail.com

Received: 30 March 2018

Accepted: 6 July 2018

ABSTRACT

In this research, reverse-phase high performance liquid chromatography using greenmobile phase utilizing short column was developed for the determination of some contraceptivedrugs such as ethinyl estradiol (EE), cyproterone acetate (CPA), gestodene (GES) andlevonorgestrel (LNG). This Zorbax SB-C18 (12.5×4.6 mm i.d., 5 μm) that commonly usedas guard column has been used instead of the expensive and conventional analytical column.The chromatographic behaviors were studied to confirm the performance of this short columnand it was found that the reciprocal value of capacity factor of each analyte was linearto micellar concentration. The optimum conditions were 0.05 mol L-1 sodium dodecylsulfate (SDS) and isopropanol (95:5, v/v) as micellar mobile phase with a flow rate of 1.0 mLmin-1. The detection wavelengths were 281, 281, 240 and 240 nm for EE, CPA, GES andLNG, respectively. A green extractant was used in this method instead of organic solventto extract the analytes. The calibration curves of each contraceptive drug were linear withR2 > 0.9990 and detection limits were 0.003, 0.06, 0.025 and 0.05 μg mL-1 for EE, CPA,GES and LNG, respectively. The proposed method was applied successfully for thedetermination of contraceptive drugs in oral contraceptive pills. The developed system isnot only a green analytical approach but also an effective and inexpensive method for thistype of analysis.

Keywords: liquid chromatography, surfactant, micellar mobile phase, short column,contraceptive drugs

Chiang Mai J. Sci. 2019; 46(1) 119

1. INTRODUCTION

Oral contraceptives are medicationsthat prevent pregnancy. They are a veryeffective method for birth control. Oralcontraceptives are pharmaceuticalformulations containing steroid hormones ina relatively small amount. Female sexhormones consist of estrogen and progestin.Therefore, oral contraceptives are hormonalpreparations that may contain combinationsof the hormones estrogen and progestinor progestin alone [1, 2].

More stable estrogens, such as ethinyl

estradiol (EE) and progestogens (orprogestins), such as levonorgestrel (LNG),gestodene (GES) and cyproterone acetate(CPA) are used more frequently for medicalpurposes. The structures of EE, CPA,GES and LNG are shown in Table 1.The prolonged use of the hormones causelong-term risks that are related to dosesand to individual susceptibility [3]. Therefore,the effective and sensitive method ofquantitative determination in pharmaceuticalpreparations is required.

Table 1. Structures and molecular weight (MW) of contraceptive drugs.

Contraceptive drugs

Ethinyl estradiol (EE)

Cyproterone acetate (CPA)

Gestodene (GES)

Levonorgestrel (LNG)

Structure MW (g mol-1)

296.403

416.940

310.430

312.446

120 Chiang Mai J. Sci. 2019; 46(1)

Various analytical methods have beendeveloped for determination of contraceptivedrugs in different matrices (includingpharmaceutical preparations [1, 4-8],environmental samples [9, 10] and biologicalsample [11-16]) such as high performanceliquid chromatography [1-5, 7, 9, 15-20],gas chromatography [12, 21], micellarelectrokinetic capillary chromatography[22], capillary liquid chromatographyand capillary electrochromatography [23].HPLC techniques are commonly used forcontraceptive drugs analysis [10].

Most HPLC systems for thedetermination of contraceptive drugs consistof an organic mobile phase (methanoland/or acetonitrile), C18 analytical column(100-250 mm length) and UV detection.The conventional analytical columns inHPLC are expensive and long, thus the analysistime is quite long. Moreover, the uses oforganic mobile phases such as methanoland/or acetonitrile, etc., are not safe. For thisreason, the traditional LC techniques usedfor the analysis of pharmaceutical consumetremendous amounts of toxic organicsolvents and consequently generate largequantities of waste. Most of them are harmfulto the environment and operators. Therefore,the green analytical method has gainedincreasing interest in pharmaceutical analysis.There are several approaches to achieve thisaim such as using eco-friendly solvents,minimizing organic solvent consumption,reducing waste generation, reducing thechromatographic separation time, etc [24-27].This proposed method implemented by usingeco-friendly mobile phase and shorteningthe analytical column length to minimizehazardous solvent consumption and wasteproduction.

In this work, SDS has been used as amicellar mobile phase with short column(C18, 12.5 mm length) instead of the

expensive and conventional analyticalcolumn. The optimized method has beenapplied to determine EE, CPA, GES andLNG in oral contraceptive pills.

2. MATERIALS AND METHODS

2.1 Reagents and ChemicalsAll chemicals and reagents used in

this work were of analytical reagent (AR)grade. Ethinyl estradiol (EE) and gestodene(GES) were purchased from Sigma (China).Cyproterone acetate (CPA) was purchasedfrom Sigma (Canada). Levonorgestrel (LNG),USP reference standard, was purchased fromUSP (Germany). Sodium dodecyl sulfate(SDS) was purchased from Carlo Erba (Italy).HPLC-grade methanol and 2-Propanol(Isopropanol, IPA) were purchased fromMerck (Germany). Water was deionized andpurified on an ultrapure water purificationsystem (Siemens Water Technologies, USA)and used to prepare all solutions.

The oral contraceptive samples consistedof 13 commercial samples (different brands,Sample No. 1-13) with three formula ofthe binary compositions such as EE+CPA,EE+GES and EE+LNG. They werepurchased from drugstores in Pathum Thaniprovince, Thailand.

2.2 Instrumentation and ChromatographicConditions

HPLC analyses were carried out makinguse of LC-30A Nexera liquid chromatograph(Shimadzu, Japan) consisting of a DGU-20A5on-line degasser, a LC-30AD pump, aCTO-20AC column oven, a SIL-30ACautosampler, a SPD-M20A diode arraydetector (DAD) and a CBM-20A systemcontroller. Data acquisition and processingwere controlled by the LabSolutions software(Shimadzu, Japan). The short column ZorbaxSB-C18 (12.5 × 4.6 mm i.d., 5 μm), AgilentTechnologies (USA), was used as an analytical

Chiang Mai J. Sci. 2019; 46(1) 121

column. The chromatographic conditionswere 0.05 mol L-1 SDS and isopropanol(95:5, v/v) as micellar mobile phase with aflow rate of 1.0 mL min-1 and an injectionvolume of 20 μL. The detection wavelengthswere 281, 281, 240 and 240 nm for EE,CPA, GES and LNG, respectively.

2.3 Standard Solution PreparationThe stock standard solutions of EE,

CPA, GES and LNG were prepared ata concentration of 500, 4000, 500 and500 μg mL-1 by accurately weighing 0.0255,0.2041, 0.0255 and 0.0250 g, respectivelyand dissolving each of them in 10.0 mLmethanol followed by dilution to 50.0 mLwith deionized water. The stock solutionswere stored in a refrigerator at 4 °C.The working standard solutions wereprepared daily by further dilution of the stocksolutions with a mobile phase and filteredthrough a 0.20 μm nylon syringe filter(Chromex Scientific, UK) before injectinginto the HPLC system.

The stock SDS solution at a concentrationof 0.5 mol L-1 was prepared by weighing72.1225 g and then adding 400 mL deionizedwater. The solution was stirring using amagnetic stirrer (Nickel-Electro Ltd., UK)for 15 min until it was dissolved. Thesolution was further diluted to 500.0 mL withdeionized water. The mobile phases wereprepared at the desired concentration bydiluting the proper amount of this stocksolution. Finally, the solutions were filteredthrough a 0.20 μm nylon membrane filter(Chromex Scientific, UK).

2.4 Sample PreparationTo evaluate the performance of the

proposed method, the commercial oralcontraceptive tablets were analyzed.

Twenty-one tablets were weighed and groundin a porcelain mortar to let it fine andhomogeneous powder. An appropriateportion of powder equivalent to the mass oftwo tablets was accurately weighed anddissolved in 2.5 mL of methanol into25.0 mL volumetric flask. This mixturewas sonicated (CT Brand, China) for 15 minand then adjusted to the volume with themobile phase. The content of the flaskwas sonicated again for 5 min to completedissolution of the drug and then centrifuged(Gemmy Industrial, Taiwan) at 4000 rpmfor 15 min. The clear supernatant wasfinally filtered through a 0.20 μm nylonsyringe filter and 20 μL aliquot was injectedinto the HPLC system.

3. RESULTS AND DISCUSSION

3.1 Preliminary StudyFor the preliminary study, the effect of

organic modifier addition on the mobilephase was investigated. Organic modifierused in this method was isopropanol.The mobile phase containing 0 and 10 %v/vof isopropanol in 0.05 mol L-1 SDS wasstudied with Zorbax SB-C18 (12.5 × 4.6 mmi.d., 5 μm) as an analytical column by injectingeach standard solution of EE, CPA, GESand LNG at concentration of 20 μg mL-1 ata flow rate of 1.0 mL min-1 and an injectionvolume of 20 μL.

The results showed that the additionof isopropanol into mobile phase affectsretention time and peak shape of the analytes.The chromatograms are shown in Figure 1.The results demonstrate that the mobilephase containing isopropanol not onlydecrease retention time but also improvethe peak symmetry. Therefore, isopropanolwas selected for the chromatographiccondition.

122 Chiang Mai J. Sci. 2019; 46(1)

Figure 1. Chromatograms of (a) EE, (b) CPA, (c) GES and (d) LNG (each concentration20 μg mL-1) using mobile phase containing 0.05 mol L-1 SDS (---) with and (−−) withoutisopropanol (10, v/v). Chromatographic condition; column: Zorbax SB-C18 (12.5 × 4.6 mmi.d., 5 μm), flow rate: 1.0 mL min-1, injection volume: 20 μL.

3.2 Chromatographic BehaviorsSince a short analytical column has been

used, the chromatographic behavior needsto confirm the performance of the system.

3.2.1 Chromatographic behavior of solutesin a micelle system

The chromatographic behavior ofsolutes in a micelle system with conventionalanalytical column C8 and C18 was reviewed[28], according to the equation 1.

1/k = C0 + C

1⋅[M] (1)

Where k is capacity factor (k = (tR - t

0)

/t0), C

0 and C

1 are formal constants. [M] is

the concentration of micelle in the mobile

phase and [M] is equal to the differencebetween total concentration of surfactant,[S], and its critical micelle concentration,CMC ([M] = [S] - CMC).

For the study of the chromatographicbehavior, concentrations of surfactant (SDS)at higher the critical micellar concentration(CMC of SDS is 8.27 mmol L-1) were studiedat 0.025, 0.05, 0.075, 0.10, 0.125 and0.15 mol L-1. The flow rate of mobilephase was 1.0 mL min-1 and the injectionvolume was 20 μL. EE, CPA and GES at aconcentration of 20 μg mL-1 and LNG at aconcentration of 5 μg mL-1 were injected intothe system. The capacity factor of eachcontraceptive drug and the chromatographicbehavior of solutes in a micelle system

Chiang Mai J. Sci. 2019; 46(1) 123

were then calculated.It was found that the reciprocal value of

capacity factor (k) of EE, CPA, GES andLNG versus the concentration of micellein the mobile phase [M] were linear, as shownin equation 2, 3, 4 and 5, respectively, andR2 > 0.99 (Figure 2 (a)), corresponding tochromatographic behavior of solutes in amicelle system.

1/kEE

= 2.4943[M] + 0.0507 (R2 = 0.9911)(2)

1/kCPA

= 1.0555[M] + 0.0168 (R2 = 0.9938)(3)

1/kGES

= 1.0505[M] + 0.0062 (R2 = 0.9931)(4)

1/kLNG

= 0.8935[M] + 0.0055(R2 = 0.9940)(5)

3.2.2 Chromatographic behavior ofsolutes in RP-HPLC system

The chromatographic behavior of solutesin RP-HPLC system with conventionalanalytical column C8 and C18 was overreviewed [29], according to the equation 6.

log k = am - m

hvbϕ (6)

Where k is capacity factor (k = (tR - t

0)

/t0), a

m and m

hvb are formal constants. ϕ is the

volume fraction of organic modifier in themobile phase.

For the study of the chromatographicbehavior of solutes in RP-HPLC system,isopropanol was mixed with SDS andvaried ratios at 0, 2.5, 5.0, 7.5, 10.0 and12.5 %v/v. The chromatographic conditionis described in Section 3.2.1. The capacityfactor of each contraceptive drug and thechromatographic behavior of the solutes inRP-HPLC system were then computed.

The results showed that the log k of EE,CPA, GES and LNG were in a linear inrelation to volume fraction (ϕ), as shownin equation 7, 8, 9 and 10, respectively, andR2 > 0.99 (Figure 2 (b)), correspondingto chromatographic behavior of solutes inRP-HPLC system.

log kEE

= -3.0253ϕ + 0.9714 (R2 = 0.9899)(7)

log kCPA

= -3.4899ϕ + 1.4316 (R2 = 0.9555)(8)

log kGES

= -2.1722ϕ + 1.4310 (R2 = 0.9695)(9)

log kLNG

= -2.0805ϕ + 1.4955(R2 = 0.9441)(10)

As has been described in Section 3.2.1and 3.2.2, the results demonstrate that itis possible to use the short column, ZorbaxSB-C18 (12.5 × 4.6 mm i.d., 5 μm), as ananalytical column for the separation ofcontraceptive drugs.

124 Chiang Mai J. Sci. 2019; 46(1)

3.3 Optimization of the ChromatographicCondition

The chromatographic conditions such ascompositions of mobile phase (organicmodifier and concentration of micellar mobilephase) and flow rate were optimized makinguse of univariate optimization. Initially,Zorbax SB-C18 (12.5 × 4.6 mm i.d., 5 mm)has been used as an analytical column at aflow rate of 1.0 mL min-1 and the injectionvolume was 20 μL.

3.3.1 Compositions of mobile phase3.3.1.1 Effect of organic modifier

As described in Section 3.1, the use ofincreased concentrations of isopropanol withreverse phase column leads to significantdecrease in retention time and alsoimprove the symmetric of peaks. Therefore,isopropanol content in mobile phase wasoptimized. The amount of the modifier wasvaried in the range of 0-12.5 %v/v. The resultsshowed that the retention time of EE, CPA,GES and LNG peaks decreased whenorganic modifier increased, as shown inFigure 3 (a). The addition of higher amountof isopropanol led to shorter retention timesand worsened the separation. Therefore,the mobile phase containing 5 %v/v of

isopropanol was selected as an optimum forthe proposed method.

3.3.1.2 Effect of concentration ofmicellar mobile phase

The concentration of surfactant (SDS)in mobile phase has a key effect onchromatographic separation. In this method,the surfactant (SDS) amounts exceeding thecritical micellar concentrations were variedin the range of 0.025-0.15 mol L-1. It wasfound that the retention time of EE,CPA, GES and LNG decreased when theconcentration of SDS increased, as clear inFigure 3 (b). Thus, SDS at a concentration of0.05 mol L-1 was chosen for the developedchromatographic method with satisfiedseparation efficiency and analytical time.

3.3.2 Flow rateThe flow rate in HPLC systems affects

the migration time of the analytes. Increasingflow rate usually cause faster elution. In thiswork, the effect of diffident flow rate at 0.6,0.8, 1.0 and 1.2 mL min-1 (Figure 3 (c)) wasexamined. The results showed that flow rateat 1.0 mL min-1 was selected for the proposedmethod. These conditions were found toachieve the complete separation within 5 min.

Figure 2. The relationship between (a) the reciprocal value of capacity factors of each analyte(1/k) and the concentration of surfactant as micelle foam in mobile phase ([M]) and (b) thelog k and the volume fraction (ϕ) of organic modifier in the mobile phase.

Chiang Mai J. Sci. 2019; 46(1) 125

3.4 Study of Some Analytical FeaturesThe linearity, limit of detection (LOD),

limit of quantification (LOQ), repeatability,reproducibility and recovery were examinedfor validation of the method.

3.4.1 LinearityThe working range of the method was

studied by performing three replicateinjections at eight standard concentrations ofEE, CPA, GES and LNG. The calibrationcurves were constructed from peak area versusconcentration of each analyte. The resultsshowed that the working range of the method

of EE, CPA, GES and LNG were 0.5-60,1-1200, 1-60 and 1-60 μg mL-1, respectivelyand R2 of each analyte were higher than0.9990 (Table 2).

3.4.2 Limit of detection (LOD) andquantification (LOQ)

The detection and quantification limitswere studied (3S/N and 10S/N, respectively).The limits of detection were 0.003, 0.06, 0.025and 0.05 μg mL-1 for EE, CPA, GES andLNG, respectively. The limits of quantificationwere 0.01, 0.20, 0.08 and 0.16 μg mL-1 forEE, CPA, GES and LNG, respectively.

Figure 3. The optimization of the chromatographic condition (a) the effect of organic modifier,(b) the effect of concentration of micellar mobile phase and (c) the effect of flow rate.Chromatographic condition; column: Zorbax SB-C18 (12.5 × 4.6 mm i.d., 5 μm), injectionvolume: 20 μL.

126 Chiang Mai J. Sci. 2019; 46(1)

The results are illustrated in Table 2.

3.4.3 Repeatability and reproducibilityTo determine method precision,

standard solutions (30 μg mL-1 of EE, GESand LNG and 600 μg mL-1 of CPA) wereinjected into the system ten times using thesame standard solution. The results areshown in Table 2. In all instances, the RSDvalues of retention time and peak arearesponse were less than 0.3%, indicatingexcellent repeatability of the assay.

Reproducibility of analysis was studiedby selecting 3 different formulation samples(Sample No. 1, Sample No. 9 and SampleNo. 11). The sample solutions were extractedwith mobile phase and then analyzed, asdescribed in Section 2.4. In all instances, the

RSD values of the amount of contraceptivedrugs in sample solutions were less than2.0% (the results were summarized inTable 2). The values of RSD obtained forthe amount of contraceptive drugs provesthe good reproducibility of the method.

3.4.4 Recovery studyRecovery study was carried out by

spiking three samples (Sample No. 1, 9and 11) with three known amounts at 50,100 and 150% of labeled amount of eachanalyte. The results showed that the recoveriesof Sample No. 1, 9 and 11 were in the rangeof 98-102% (Table 2). Therefore, therecovery percentage proved that the methodwas sufficiently accurate within the desiredrange.

Table 2. Some analytical features of the proposed method.

Analytical featuresLinearity

- Range (μg mL-1)- Slope- Intercept- R2

LOD (3S/N)(μg mL-1)LOQ (10S/N)(μg mL-1)Repeatability(%RSD, n=10)

- Retention time- Peak area

Reproducibility(%RSD, n=3)

- Sample No. 1- Sample No. 9- Sample No. 11

Recovery* (%)- Sample No. 1- Sample No. 9- Sample No. 11

EE

0.5-6056992-72690.9998

0.003

0.01

0.260.04

0.451.250.94

98.79-99.3699.76-100.4999.49-101.27

CPA

1-120025851

-2339860.9995

0.06

0.20

0.120.05

2.0--

98.17-99.52--

GES

1-6033314-268880.9993

0.025

0.08

0.120.17

-0.24

-

-98.02-100.85

-

LNG

1-6029359-127860.9995

0.05

0.16

0.150.14

--

0.35

--

99.25-101.30* spiked each of analyte with three concentration levels into three sample solutions.

Chiang Mai J. Sci. 2019; 46(1) 127

3.5 Analysis of SamplesTo demonstrate the applicability of the

proposed method, it has been used for theanalysis of the commercial oral contraceptives.The sample solutions were prepared andthen analyzed, as described in Section 2.4.Each sample was determined in triplicate.

The chromatograms of the standard atoptimal condition are shown in Figure 4.The amounts of each analyte found in thesamples and %label amount are summarizedin Table 3. The results indicated that allsamples were within the USP range of90.0-110.0% of the labeled amount [30].

Figure 4. Chromatograms of (a) EE and CPA, (b) EE and GES, and (d) EE and LNG.Chromatographic condition; column: Zorbax SB-C18 (12.5 × 4.6 mm i.d., 5 μm), mobilephase: 0.05 mol L-1 SDS and isopropanol (95:5, v/v), flow rate: 1.0 mL min-1, injection volume:20 μL, detection wavelengths: 281, 281, 240 and 240 nm for EE, CPA, GES and LNG.

128 Chiang Mai J. Sci. 2019; 46(1)

Table 3. Analysis of EE, CPA, GES and LNG quantity in oral contraceptive pills.

Sample No.

1

2

3

4

5

6

7

8

9

10

11

12

13

Analytes

EECPAEECPAEECPAEECPAEECPAEECPAEE

GESEE

GESEE

GESEE

GESEE

LNGEE

LNGEE

LNG

Labeled (μg/tb)

35200035

200035

200035

200035

200035

20001560156020752075301503015030150

Average found±SD*

(μg/tb)35.39±0.161961±40

34.43±0.421957±1

35.09±0.252034±13

35.06±0.332022±29

34.85±0.081974±29

34.68±0.261974±34

15.20±0.2761.18±0.6815.27±0.0460.48±0.3219.66±0.2575.10±0.1819.78±0.3774.78±0.2029.99±0.28150.4±0.530.16±0.42148.8±1.130.16±0.31152.6±1.0

Average % labelamount±SD*

101.1±0.598.0±2.098.4±1.297.9±0.1100.3±0.7101.8±0.7100.2±0.9101.1±1.599.6±0.298.7±1.599.1±0.798.7±1.7101.3±1.8102.0±1.1101.8±0.3100.8±0.598.3±1.2100.1±0.298.9±1.999.7±0.3100.0±0.9100.3±0.4100.5±1.499.2±0.7100.5±1.0101.8±0.7

* SD = standard deviation (n=3).

This proposed method with ashort column (12.5 mm length) and anenvironmental friendly mobile phasewas compared to the published HPLCmethods for the determination of somecontraceptive drugs in oral contraceptives[2, 4, 7, 19, 31-32]. The parameters thatwere compared include chromatographicconditions, some analytical features and

solvent consumption as shown in Table 4.The advantage of this proposed methodover other HPLC methods is the reductionof waste generation (more than 10 times)with short analysis time. Moreover, noorganic solvent was used in the samplepreparation process and the cost of the shortcolumn is cheaper than the conventionalcolumn (125-250 mm length).

Chiang Mai J. Sci. 2019; 46(1) 129

Tab

le 4

. Com

para

tive

revi

ew o

f so

me

cont

race

ptiv

e dr

ugs

anal

ysis

in o

ral c

ontr

acep

tives

by

LC

tec

hniq

ues.

Yea

r

2004

2008

2013

2013

2016

2016

The

purp

osed

met

hod

Col

umn

C18

(150

×4.6

mm

,5

μm)

C18

(150

×4.6

mm

,4

μm)

C18

(250

×4.6

mm

,5

μm)

C18

(125

×4.0

mm

,5

μm)

C18

(150

×4.6

mm

,5

μm)

C18

(150

×4.6

mm

,5

μm)

C18

(12.

5×4.

6 m

m,

5 μm

)

Mob

ile p

hase

met

hano

l:wat

er(8

0:20

)

acet

onit

rile

:m

etha

nol:w

ater

(30:

20:5

0)ac

eton

itri

le:

wat

er (

80:2

0)

acet

onit

rile

:w

ater

(50

:50)

acet

onit

rile

:w

ater

(50

:50)

acet

onit

rile

:m

etha

nol:w

ater

(60:

15:2

5)0.

05 m

ol L

-1

SDS:

isop

ropa

nol

(95:

5)

Flo

w r

ate

(mL

min

-1)

1.0

1.0

1.0

1.0

1.0

1.0

1.0

Det

ectio

na

UV

UV

UV

Fluo

resc

ence

UV

UV

UV

UV

Ana

lysi

stim

e(m

in)

6 20 7 7 8 4 5

Solv

ent

cons

umpt

ionb

(mL

)48 30 48 30 30 45 3

Ana

lyte

Eth

inyl

est

radi

olG

esto

dene

Eth

inyl

est

radi

olL

evon

orge

stre

lG

esto

dene

Eth

inyl

est

radi

olL

evon

orge

stre

l

Eth

inyl

est

radi

olD

rosp

ireno

ne

Eth

inyl

est

radi

olD

rosp

ireno

neG

esto

dene

Lev

onor

gest

rel

Eth

inyl

est

radi

olL

evon

orge

stre

l

Eth

inyl

est

radi

olC

ypro

tero

neG

esto

dene

Lev

onor

gest

rel

Lin

eari

ty( μ

g m

L-1)

5-80

10-2

00

2.4-

6012

-300

9-16

04-

2520

-125

0.1-

1015

-150

0

0.62

2-1.

866

60.0

-180

.01.

512-

4.53

63.

0-9.

02-

141-

70

0.5-

601-

1200

1-60

1-60

LOD

( μg

mL

-1)

0.8c

2.3c

- - -0.

032c

0.06

6c

0.02

c

4.88

c

0.02

8c

9.50

0c

0.13

2c

0.76

3c

0.03

d

0.84

d

0.00

3e

0.06

e

0.02

5e

0.05

e

Ref

.

[7]

[2]

[4]

[19]

[31]

[32] -

a U

V:

Ultr

avio

let

dete

ctio

n, M

S: M

ass

spec

trom

etry

b O

rgan

ic s

olve

nt i

n m

obile

pha

se p

er h

our

c T

he l

imit

of d

etec

tion

(LO

D)

was

an

estim

atio

n of

3.3

(s/S

); w

here

s i

s th

e st

anda

rd d

evia

tion

of y

-int

erce

pt a

nd S

is

the

slop

e of

the

cal

ibra

tion

curv

e.d

The

lim

it o

f de

tect

ion

(LO

D)

was

an

esti

mat

ion

of 3

(s/S

); w

here

s i

s th

e st

anda

rd d

evia

tion

of

y-in

terc

ept

and

S is

the

slo

pe o

f th

e ca

libra

tion

curv

e.e

The

lim

it of

det

ectio

n (L

OD

) w

as a

n es

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ion

of 3

S/N

.

130 Chiang Mai J. Sci. 2019; 46(1)

4. CONCLUSIONS

This work proposed the use of a shortcolumn (Zorbax SB-C18) as an analyticalcolumn with an environmental friendlymobile phase for the determination of binarymixer of contraceptive drugs such as EE,CPA, GES and LNG. Under the optimumcondition, the proposed method wassuccessfully applied for the determinationof contraceptive drugs in commercial oralcontraceptive pills. The results indicatedthat all samples were within the requirementof USP. The proposed method present agreen analytical liquid chromatography hasthe potential of cost effective analysis, reducedchemical consumption and minimum wastegeneration.

ACKNOWLEDGEMENTS

The authors gratefully acknowledgethe nancial support provided by ThammasatUniversity Research Fund under the TUResearch Scholar, Contract No. 1/24/2557.The authors are also thankful to the Centerof Scientific Equipment for AdvancedResearch (TU-CSEAR), the Central ScienticInstrument Center (CSIC) and Departmentof Chemistry, Faculty of Science andTechnology, Thammasat University, forfacility and instrument support throughoutthis research.

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