**3. Results and discussion**

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

Effects of derivatization time and concentration of DNPH for MPA analysis were studied. The DNPH concentrations used were 12.5, 25, 75 and 100 mg/mL. MPA (100 ng/mL) was used to react with various concentrations of DNPH in the ratio of 1 : 1 at room temperature and their reaction times were also done up to 180 min (Fig. 3). It was found that 75 mg/mL of DNPH gave the highest peak area within 120 min derivatization time. In this study, the derivatization time of 30 min wasenough, since the signal output was not significantly different from that of 120 min derivatization.

Application of HPLC Analysis of Medroxyprogesterone Acetate in Human Plasma 223

the results for intra-day and inter-day precision were 5.05 and 11.5 %RSD, respectively. The intra-day accuracy ranged from 102.2 - 109.5%. The average recovery was found to be 102.9 + 4.4%. It was found that this electrochemical detection was considerably selective and gave a working range of 1.0 - 10.0 ng/mL with linear regression: y = (3.17 + 0.12) x – (0.67 + 0.03), r2

**Figure 4.** Effect of temperatures on the derivatization reaction of DNPH (75 mg/mL) and MPA (100

**Figure 5.** Effect of applied voltammetric potentials on the peak areas of MPA- and P-DNPH derivatives.

= 0.9985 for *n* = 5 with %RSD = 2.34 – 7.96.

ng/mL) between 5 min and 30 min.

**Figure 3.** Effects of reaction times and concentrations on the derivatization of DNPH with 100 ng/mL MPA at room temperature.

The effect of temperature for 30 min derivatization was also investigated using 100 ng/mL MPA and 75 mg/mL DNPH in acidic solution. There seemed to be no effect of temperature, except a linear increase of peak area when the reaction was performed at room temperature (Fig. 4). Thus, the reaction was rapidly completed at room temperature.

Concerning on RP-HPLC-AD analysis, the optimization conditions of the HPLC-AD for MPA-DNPH analysis were then investigated. Effects of pH (3-6) and buffer concentration (10-40 mM) on mobile phase (ACN : phosphate buffer) were carried out. It was found that the optimum buffering system was at pH 3.0 and 30 mM phosphate buffer (data not shown). In addition, the ratios of organic solvents were investigated and a good separation was obtained with the mobile phase of ACN : MeOH : 30 mM phosphate buffer (pH 3.0) at the ratio of 39 : 39 : 22 by volume.

Figure 5 shows the hydrodynamic voltammogram (HDV) of MPA- and P-DNPH derivatives. The optimum potential that gave the highest peak area was around 0.8-0.9 V for both derivatized products, and 0.85 V was used in this study.

For method validation, the method limits for detection and quantitation were found to be 0.2 and 1.0 ng/mL with lower 15% precision and 80 - 120% accuracy, respectively. In Table 1, the results for intra-day and inter-day precision were 5.05 and 11.5 %RSD, respectively. The intra-day accuracy ranged from 102.2 - 109.5%. The average recovery was found to be 102.9 + 4.4%. It was found that this electrochemical detection was considerably selective and gave a working range of 1.0 - 10.0 ng/mL with linear regression: y = (3.17 + 0.12) x – (0.67 + 0.03), r2 = 0.9985 for *n* = 5 with %RSD = 2.34 – 7.96.

222 Chromatography – The Most Versatile Method of Chemical Analysis

different from that of 120 min derivatization.

MPA at room temperature.

ratio of 39 : 39 : 22 by volume.

Effects of derivatization time and concentration of DNPH for MPA analysis were studied. The DNPH concentrations used were 12.5, 25, 75 and 100 mg/mL. MPA (100 ng/mL) was used to react with various concentrations of DNPH in the ratio of 1 : 1 at room temperature and their reaction times were also done up to 180 min (Fig. 3). It was found that 75 mg/mL of DNPH gave the highest peak area within 120 min derivatization time. In this study, the derivatization time of 30 min wasenough, since the signal output was not significantly

**Figure 3.** Effects of reaction times and concentrations on the derivatization of DNPH with 100 ng/mL

The effect of temperature for 30 min derivatization was also investigated using 100 ng/mL MPA and 75 mg/mL DNPH in acidic solution. There seemed to be no effect of temperature, except a linear increase of peak area when the reaction was performed at room temperature

Concerning on RP-HPLC-AD analysis, the optimization conditions of the HPLC-AD for MPA-DNPH analysis were then investigated. Effects of pH (3-6) and buffer concentration (10-40 mM) on mobile phase (ACN : phosphate buffer) were carried out. It was found that the optimum buffering system was at pH 3.0 and 30 mM phosphate buffer (data not shown). In addition, the ratios of organic solvents were investigated and a good separation was obtained with the mobile phase of ACN : MeOH : 30 mM phosphate buffer (pH 3.0) at the

Figure 5 shows the hydrodynamic voltammogram (HDV) of MPA- and P-DNPH derivatives. The optimum potential that gave the highest peak area was around 0.8-0.9 V for

For method validation, the method limits for detection and quantitation were found to be 0.2 and 1.0 ng/mL with lower 15% precision and 80 - 120% accuracy, respectively. In Table 1,

(Fig. 4). Thus, the reaction was rapidly completed at room temperature.

both derivatized products, and 0.85 V was used in this study.

**Figure 4.** Effect of temperatures on the derivatization reaction of DNPH (75 mg/mL) and MPA (100 ng/mL) between 5 min and 30 min.

**Figure 5.** Effect of applied voltammetric potentials on the peak areas of MPA- and P-DNPH derivatives.


Application of HPLC Analysis of Medroxyprogesterone Acetate in Human Plasma 225

**Table 1.** Intra-day and inter-day precision and method recovery of MPA-DNPH derivative. <sup>a</sup>*n* = 5

*bwithin three consecutive days cMean of three determinations RSD, relative standard deviation*

Since the method sensitivity of MPA analysis was the main objective of this study, it has been shown that the clean-up step, using the SPE method for MPA spiked in human plasma sample, was necessary probably due to both the reagent used and the sample interference matrices (Fig. 6, a-d). The solution of MPA-DNPH derivative products with excess DNPH was not clean enough, so it must be added through the Sep-pak cartridge before being separated on the HPLC-AD system. Without the clean-up step, there might be trouble for the HPLC column used, which may affect the analytical sensitivity as well. This is the first report of the use of DNPH as an electroactive labeling reagent for MPA analysis. Development and validation of the HPLC-AD of the derivatized product were then carried out. The MPA-DNPH was completely separated from the I.S. within a suitable analysis time (Fig. 6, a-d).

Intra-day RSD (%)

**Table 1.** Intra-day and inter-day precision and method recovery of MPA-DNPH derivative.

Inter-dayb RSD (%)

1.0 7.76 15.4 107.3 4.0 5.06 9.62 98.55 8.0 2.35 9.55 102.9 Mean + SD 5.05 + 2.7 11.5 + 3.4 102.9 + 4.4

Since the method sensitivity of MPA analysis was the main objective of this study, it has been shown that the clean-up step, using the SPE method for MPA spiked in human plasma sample, was necessary probably due to both the reagent used and the sample interference matrices (Fig. 6, a-d). The solution of MPA-DNPH derivative products with excess DNPH was not clean enough, so it must be added through the Sep-pak cartridge before being separated on the HPLC-AD system. Without the clean-up step, there might be trouble for the HPLC column used, which may affect the analytical sensitivity as well. This is the first report of the use of DNPH as an electroactive labeling reagent for MPA analysis. Development and validation of the HPLC-AD of the derivatized product were then carried out. The MPA-DNPH was completely separated from the I.S. within a suitable analysis time

Recoveryc (%)

Concentrationa (ng/mL)

*bwithin three consecutive days cMean of three determinations RSD, relative standard deviation*

<sup>a</sup>*n* = 5

(Fig. 6, a-d).

Application of HPLC Analysis of Medroxyprogesterone Acetate in Human Plasma 227

Table 2. It is, however, satisfactory for pharmacokinetic study of trace MPA in plasma sample, especially in the case of contraceptive administration with very low dose. Since it is rapid procedure and inexpensive, this technique is suitable for routine analysis of the MPA

A specific and sensitive method is presented for the analysis of medroxyprogesterone acetate (MPA) in human plasma using reversed phase high-performance liquid chromatography (HPLC) with amperometric detection. The blood sample spiked with trace amount of MPA was cleaned up to remove natural interfering matrices by solid-phase extraction (SPE). The MPA extract was then derivatized with 2,4-dinitrophenylhydrazine (DNPH) as an electroactive agent. The MPA-DNPH derivative was re-extracted using SPE prior to analysis by reversed phase HPLC. Quantitative analysis of the MPA-DNPH using prednisolone-DNPH as an internal standard were optimized on a Hypersil ODS column using acetonitrile : methanol : 30 mM phosphate buffer, pH 3.0 (39 : 39 : 22, v/v/v) as mobile phase at a flow-rate of 1.0 mL/min. It was found that the method was selective and gave linear calibration curve for a concentration range of 1.0 - 10.0 ng/mL for 2 mL spiked plasma samples. The relative standard deviation (RSD) of inter-day precision for a period of three validation days was 11.5 + 3.4% for all concentration used. The RSD of intra-day precision (n = 5) was 5.05 + 2.7% with accuracy (n = 5) of 102.3 + 7.4%. The average recovery was found to be 102.9 + 4.4%. The correlation coefficient of the calibration curve was 0.9985. The limits of detection and quantitation were found to be 0.2 and 1.0 ng/mL, respectively. Using DNPH as a derivatizing agent can enhance both selectivity and sensitivity of MPA in plasma and is

content in most pharmaceutical products and blood samples.

**4. Conclusion** 

suitable for routine analysis.

ACN: Acetonitrile

HCl: Hydrochloric acid

 chemiluminescence I.S.: Internal standard

LOD: Limit of detection LOQ: Limit of quantitation

MeOH: methanol

Depot-MPA: Depot-medroxyprogesterone acetate

GC-ECD: Gas chromatography-electron capture detection GC-MS: Gas chromatography-mass spectrometry

HPLC-UV: High-performance liquid chromatography with ultraviolet detection HPLC-PO-Cl: High-performance liquid chromatography with peroxyoxalate

LC-MS/MS: Liquid chromatography-mass spectrometry/mass spectrometry

DNPH: 2,4-Dinitrophenylhydrazine

HDV: Hydrodynamic voltammogram

**5. Abbreviations** 

**Figure 6.** Chromatogram obtained from (a) blank reagent. (b) standard solution of (1) MPA-DNPH (10 ng/mL) and (2) P-DNPH as I.S. (c) blank of blood plasma sample, (d) blood plasma spiked with (1) MPA-DNPH (10 ng/mL) and (2) P-DNPH as I.S. (inset, (1) MPA-DNPH, 1 ng/mL).


**Table 2.** Some analytical methods and limits of detection of MPA analysis in plasma samples.

*aelectron capture detection of MPA derivative* 

*bmass spectrometric detection of MPA derivative* 

*ctandem mass spectrometry* 

*dultraviolet (254 nm) detection of MPA* 

*e peroxyoxalate chemiluminescence (*λ*ex/*λ*em : 480/570 nm) detection of MPA derivative* 

*f voltammetric (0.85 V) detection of MPA-DNPH derivative* 

*LOD, limit of detection*

The plasma sample size used in this study was still rather high (2 mL) for clinical aspects when compared with other reports [11,13,14], giving LOD and LOQ of 0.2 and 1.0 ng/mL, respectively. Thus, the developed method with electrochemical detection has sufficiently high sensitivity when compared with other methods, except for the tandem MS, as shown in Table 2. It is, however, satisfactory for pharmacokinetic study of trace MPA in plasma sample, especially in the case of contraceptive administration with very low dose. Since it is rapid procedure and inexpensive, this technique is suitable for routine analysis of the MPA content in most pharmaceutical products and blood samples.
