**1. Introduction**

216 Chromatography – The Most Versatile Method of Chemical Analysis

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## **1.1. Steroid hormones and hormonal contraceptive drugs**

Generally, progestogens, i.e. progesterone in combination with oestradiol esters, are used primarily in the treatment of menstrual irregularities to maintain endangered pregnancies. Their derivatives are orally active medroxyprogesterone acetate including hydroxyprogesterone caproate, chlormadinone acetate, megestrol acetate. Naturally occurring glucocorticoids are often used in medicine such as cortisone, hydrocortisone, prednisone and prednisolone (Fig. 1). Depot-medroxyprogesterone acetate (Depot-MPA) is commercially available and commonly known as a hormonal contraception used by women. It is a synthetic steroid hormone, which mimics natural progesterone, but its contraceptive activity has been shown to be about 30 times higher. Depot-MPA is widely used by intramuscular administration for long-term contraception [1-3].

Research concerning its pharmacokinetics has been performed in order to improve the hormonal activity of the drug formulation [2,4]. This will help to standardize the dosage of the drug.

## **1.2. Determination of MPA in hormonal contraceptive drugs**

Analysis of MPA in plasma or serum has been performed to study its pharmacokinetics and to monitor its residual levels in cancer patients after oral or intramuscular administration [4,5]. Numerous recent researches for this analysis have been reported concerning the method sensitivity and selectivity in association with various instruments including GC, HPLC and others [6-15]. However, each of which had their advantages and disadvantages. Therefore, development and validation of the method for MPA analysis in spiked blood plasma using internal standard by HPLC method with specific derivatizing agents and

clean-up by solid-phase extraction have been carried out. The method sensitivity obtained is suitable for pharmacokinetics study of MPA.

Application of HPLC Analysis of Medroxyprogesterone Acetate in Human Plasma 219

considerably inconvenient for routine analysis due to the high instrument cost. The HPLC with amperometric detector (AD) would be a highly sensitive tool. From the molecular structure, MPA is a ketone steroid in which the ketone group can undergo reduction. Since the reduction is normally easily interfered by oxygen molecule dissolved in mobile phase, this may be a problem [15]. Hydrazines including 2,4-dinitrophenyl hydrazine (DNPH) have been introduced as an electroactive labeling reagent for carbonyl compounds [16]. Until now there is no report of the derivatization of MPA with DNPH (Fig. 2). From the chemical structures condensation reaction can occur between the keto group of MPA and DNPH to give the MPA-hydrazone which can be detected by HPLC-AD. The optimum conditions of DNPH derivatization with MPA were studied. Therefore, development and validation of the method for MPA analysis in spiked blood plasma using prednisolone (P) as internal standard (I.S.) by HPLC-AD after derivatization with DNPH and clean-up by solidphase extraction have been carried out. The method sensitivity obtained is suitable for

**Figure 2.** Derivatizing reaction between 2,4-dinitrophenylhydrazine and ketosteroids giving their

Medroxyprogesterone acetate (MPA), 99.27%, used as working standard was obtained from A.N.B. Laboratory Co. Ltd. (Thailand). Predinisolone was an analytical reagent (A.R.) grade from Sigma (Germany). 2,4-Dinitrophenylhydrazine (Fluka, Switzerland) was also of A.R. grade. Methanol (MeOH, Merck, Germany), acetonitrile (ACN, Merck, Germany), dipotassium hydrogen phosphate-3-hydrate (BDH, England) and potassium dihydrogen phosphate (BDH, England) were of HPLC grade. Other chemicals used were also A.R. grade

Standard stock solution (500 mg/mL) of MPA was prepared in methanol. Dilute standard solutions of MPA were also prepared in methanol. The 2,4-dinitrophenylhydrazine stock solution used as a labeling reagent (1,000 mg/mL) was prepared by dissolving the hydrazine in 1.2 mL concentrated HCl prior to adjusting the final volume to 25 mL with methanol.

Stock phosphate solution (2 M) was prepared from sodium dihydrogen phosphate monohydrate (NaH2PO4.H2O) in deionized water. A buffer solution of 30 mM potassium

**2. HPLC-AD analysis of MPA in human plasma: A case study** 

C R2

ketosteroid

O

+

R1

H

O2N

NH N

NO2

ketosteroid-2,4-dinitrophenylhydrazone

R2

R1

+


ketosteroid-2,4-dinitrophenylhydrazones as the derivatized products.

**2.1. Reagents, standard solution and plasma sample** 

including hydrochloric acid (Merck, Germany).

pharmacokinetic study of MPA.

O2N

NH NH2

NO2

2,4-dinitrophenylhydrazine

**Figure 1.** Chemical structure of medroxyprogesterone acetate and prednisolone

Normally, the levels of MPA found in plasma range from 1.75 to 9.0 ng/mL [5]. Formerly, the measurement of MPA in a plasma sample was mostly carried out by radioimmunoassay (RIA) [6]. Trace analysis of MPA in dog plasma was achieved using cyclohexane extraction, followed by heptafluorobutylic anhydride derivatization and gas chromatography with electron capture detector (GC-ECD). However, this procedure could not be applied for MPA analysis in human plasma due to its high matrix interference. Attempts have been made to optimize the methods for MPA analysis using small amounts of plasma samples [7,8]. Quantitative analysis of MPA in plasma by HPLC with ultraviolet (UV) detection was also carried out, but did not give satisfactory detection limit [9,10]. The high sensitivity of the GC-MS has also been used to monitor the MPA level in human serum [11]. This procedure used trifluoroacetic anhydride derivatization of the extracted portion after SPE and gave the detection limit down to 0.5 ng/mL.

Although, HPLC-UV analysis of MPA in plasma has been performed, but its sensitivity was about 5 ng/mL [12]. In order to enhance both selectivity and sensitivity, recent method development and validation of MPA analysis has been focused on HPLC separation with various detection systems. HPLC with chemiluminescence detection was used to trace MPA in serum via 4-(N,N-dimethylaminosulphonyl) -7-hydrazino-2,1,3-benzoxadiazole as a fluorogenic agent [13]. Recently, MPA analysis in plasma sample has been conducted by liquid chromatography-electrospray ion trap mass spectrometry (LC-MS/MS) after liquid phase extraction and this gave 10 times higher sensitivity than GC-MS [11,14].

From these reviews, both GC-ECD and HPLC-UV did not give sensitivity high enough for MPA analysis in pharmacokinetic studies. In clinical aspects, the RIA method has a rather high sensitivity but sometimes gives positive result due to metabolite interference. Both GC-MS and LC-MS/MS are methods of choice with high selectivity and sensitivity, but may be considerably inconvenient for routine analysis due to the high instrument cost. The HPLC with amperometric detector (AD) would be a highly sensitive tool. From the molecular structure, MPA is a ketone steroid in which the ketone group can undergo reduction. Since the reduction is normally easily interfered by oxygen molecule dissolved in mobile phase, this may be a problem [15]. Hydrazines including 2,4-dinitrophenyl hydrazine (DNPH) have been introduced as an electroactive labeling reagent for carbonyl compounds [16]. Until now there is no report of the derivatization of MPA with DNPH (Fig. 2). From the chemical structures condensation reaction can occur between the keto group of MPA and DNPH to give the MPA-hydrazone which can be detected by HPLC-AD. The optimum conditions of DNPH derivatization with MPA were studied. Therefore, development and validation of the method for MPA analysis in spiked blood plasma using prednisolone (P) as internal standard (I.S.) by HPLC-AD after derivatization with DNPH and clean-up by solidphase extraction have been carried out. The method sensitivity obtained is suitable for pharmacokinetic study of MPA.

**Figure 2.** Derivatizing reaction between 2,4-dinitrophenylhydrazine and ketosteroids giving their ketosteroid-2,4-dinitrophenylhydrazones as the derivatized products.

## **2. HPLC-AD analysis of MPA in human plasma: A case study**

### **2.1. Reagents, standard solution and plasma sample**

218 Chromatography – The Most Versatile Method of Chemical Analysis

suitable for pharmacokinetics study of MPA.

detection limit down to 0.5 ng/mL.

clean-up by solid-phase extraction have been carried out. The method sensitivity obtained is

Normally, the levels of MPA found in plasma range from 1.75 to 9.0 ng/mL [5]. Formerly, the measurement of MPA in a plasma sample was mostly carried out by radioimmunoassay (RIA) [6]. Trace analysis of MPA in dog plasma was achieved using cyclohexane extraction, followed by heptafluorobutylic anhydride derivatization and gas chromatography with electron capture detector (GC-ECD). However, this procedure could not be applied for MPA analysis in human plasma due to its high matrix interference. Attempts have been made to optimize the methods for MPA analysis using small amounts of plasma samples [7,8]. Quantitative analysis of MPA in plasma by HPLC with ultraviolet (UV) detection was also carried out, but did not give satisfactory detection limit [9,10]. The high sensitivity of the GC-MS has also been used to monitor the MPA level in human serum [11]. This procedure used trifluoroacetic anhydride derivatization of the extracted portion after SPE and gave the

Although, HPLC-UV analysis of MPA in plasma has been performed, but its sensitivity was about 5 ng/mL [12]. In order to enhance both selectivity and sensitivity, recent method development and validation of MPA analysis has been focused on HPLC separation with various detection systems. HPLC with chemiluminescence detection was used to trace MPA in serum via 4-(N,N-dimethylaminosulphonyl) -7-hydrazino-2,1,3-benzoxadiazole as a fluorogenic agent [13]. Recently, MPA analysis in plasma sample has been conducted by liquid chromatography-electrospray ion trap mass spectrometry (LC-MS/MS) after liquid

From these reviews, both GC-ECD and HPLC-UV did not give sensitivity high enough for MPA analysis in pharmacokinetic studies. In clinical aspects, the RIA method has a rather high sensitivity but sometimes gives positive result due to metabolite interference. Both GC-MS and LC-MS/MS are methods of choice with high selectivity and sensitivity, but may be

phase extraction and this gave 10 times higher sensitivity than GC-MS [11,14].

**Figure 1.** Chemical structure of medroxyprogesterone acetate and prednisolone

Medroxyprogesterone acetate (MPA), 99.27%, used as working standard was obtained from A.N.B. Laboratory Co. Ltd. (Thailand). Predinisolone was an analytical reagent (A.R.) grade from Sigma (Germany). 2,4-Dinitrophenylhydrazine (Fluka, Switzerland) was also of A.R. grade. Methanol (MeOH, Merck, Germany), acetonitrile (ACN, Merck, Germany), dipotassium hydrogen phosphate-3-hydrate (BDH, England) and potassium dihydrogen phosphate (BDH, England) were of HPLC grade. Other chemicals used were also A.R. grade including hydrochloric acid (Merck, Germany).

Standard stock solution (500 mg/mL) of MPA was prepared in methanol. Dilute standard solutions of MPA were also prepared in methanol. The 2,4-dinitrophenylhydrazine stock solution used as a labeling reagent (1,000 mg/mL) was prepared by dissolving the hydrazine in 1.2 mL concentrated HCl prior to adjusting the final volume to 25 mL with methanol.

Stock phosphate solution (2 M) was prepared from sodium dihydrogen phosphate monohydrate (NaH2PO4.H2O) in deionized water. A buffer solution of 30 mM potassium

dihydrogen phosphate (KH2PO4), pH 3.0 was made by dissolving KH2PO4 in deionized water and adjusting to pH 3.0 with 6 M orthophosphoric acid (H3PO4). Mobile phase was prepared and used to optimize the separation of the mixture of MPA-DNPH and P-DNPH derivatives by amperometric detection (HPLC-AD). The suitable solvent system was composed of ACN : MeOH : 30 mM KH2PO4 (pH 3.0) in the ratio of 39 : 39 : 22 by volume according to Snyder et al. [17]. The mobile phase was filtered through nylon membrane (0.45 μm, 47 mm) and degassed for 15 min in ultrasonic bath before use. Sep-pak C18 cartridge, 100 mg (Waters Associates, U.S.A.) was used.

Application of HPLC Analysis of Medroxyprogesterone Acetate in Human Plasma 221

The clean up method using solid-phase extraction (SPE) was found to be a critical step for plasma sample both before and after derivatization with DNPH. Spiked standard plasma (MPA 6 ng/mL) was extracted by Sep-pak C18 cartridge using a mixture of MeOH : H2O as extraction solvent. The C18 cartridge was first washed with 2 mL MeOH and followed by 2 mL water. The spiked plasma (2 mL) was added into the cartridge and then washed with 2 mL water, followed by 250 mL 50%(v/v) MeOH (3x), and the final elution was made with 1 mL MeOH. The MPA extract portion was kept for derivatizing with DNPH. The solution of DNPH containing 0.1 mg was added into the MPA extract portion and allowed to stand for 30 min, followed by 0.9 mL water and 0.2 mL solution of P-DNPH (10 ng/mL). The sample of the derivative mixture was then introduced into the cartridge followed by 3 times rinsing with 1 mL 50%(v/v) MeOH. The cartridge containing MPA- and P-DNPH products was washed by 250 mL 50%(v/v) MeOH (3x) and again allowed to stand for 15 min before elution by 1 mL MeOH. The elute was then dried over a stream of N2, redissolved in 60 mL MeOH, then 40 mL MeOH added and 60 mL was injected into the

The separation of both MPA- and P-DNPH derivatives was carried out on Hypersil ODS column by HPLC-AD (0.85 V) using an electrolyte solvent system as mobile phase with a flow-rate of 1 mL/min. The mobile phase of ACN : MeOH : 30 mM KH2PO4 (pH 3.0) (30 : 39 : 22, v/v/v) was used in this experiment after optimization of buffer concentration, pH and organic modifier. Both MPA standard solution and MPA spiked plasma sample were

Various concentrations of MPA spiked in a plasma sample with 10 ng/mL P-DNPH as I.S. were extracted by Sep-pak C18 cartridge, and run by HPLC-AD (0.85 V) [20,21]. Limit of detection (LOD) is the concentration of MPA giving a peak height 3 times the baseline noise (3SD) and the limit of quantitation (LOQ) is defined as 10SD. The standard curve was obtained from the concentrations of 0.5, 1, 2, 4, 6, 8 and 10 ng/mL. The accuracy and precision including recovery were determined at three concentration levels (1, 4 and 8

For method development, studies on derivatization of some hydrazines with MPA had been carried out in acidic solution [15]. It was found that DNPH is a suitable reagent. Many internal standards having similar core structure which is derivatized with DNPH were also investigated by the same manner [19]. Prednisolone was found to be the most suitable ketosteroid and its derivatized solid product was prepared. It was eluted along with the

ii. Solid-phase extraction of MPA-DNPH derivative

iii. Analysis of MPA-DNPH by RP-HPLC-AD

ng/mL) of the spiked plasma standard.

**3. Results and discussion** 

prepared at the concentrations of 0.5, 1, 2, 4, 6 and 8 ng/mL.

iv. Method sensitivity of MPA analysis in spiked plasma sample

MPA-DNPH and was used as I.S. throughout the experiment.

HPLC system.

Human blood plasma samples were kindly obtained from the Blood Bank, Srinagarin Hospital, Khon Kaen University, Khon Kaen, Thailand.

## **2.2. Instruments and apparatus**

HPLC system (Perkin Elmer, U.S.A.) used in this study included LC200 HPLC pump with electrochemical detector (Coulochem II, ESA, U.S.A.). The standard analytical cell (ESA 5011, U.S.A.) consisted of porous graphite working electrode, palladium reference electrode and platinum counter electrode. The analytical column used was stainless steel tube packed with Hypersil ODS, 5 μm particle size (125 x 4.0 mm i.d., Agilent, U.S.A.). Integrator model1022 (Perkin Elmer, U.S.A.) was used. UV-Visible spectrophotometer (Cecil 3000, England) was also used. Analytical balance (AE 200) and pH meter (Delta 350) were from Mettler (U.S.A.). SPE manifold-12 place vacuum manifold (Lida, Germany) was used. Autopipette (Eppendorf, Germany) with volume adjustments was used throughout the experiment.
