**4. Conclusion**

226 Chromatography – The Most Versatile Method of Chemical Analysis

**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)

Instrument Extraction method Sample size (mL) LOD (ng/mL) Reference GC-ECDa liquid phase 4 1-5 [7,8] GC-MSb solid phase 1 0.5 [11]

HPLC-UVd solid phase 2 4 [9] HPLC-PO-Cle liquid phase 0.1 9 [13]

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

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

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

liquid phase 1 0.05 [14]

solid phase 2 0.2 This work

MPA-DNPH (10 ng/mL) and (2) P-DNPH as I.S. (inset, (1) MPA-DNPH, 1 ng/mL).

LC-MS/MSc

HPLC-ADf

*e*

*f*

*ctandem mass spectrometry* 

*LOD, limit of detection*

*aelectron capture detection of MPA derivative bmass spectrometric detection of MPA derivative* 

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

*dultraviolet (254 nm) detection of 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 suitable for routine analysis.
