**2.3. Method development and validation**

## i. Derivatization of MPA with DNPH

Study on the hydrazine derivatization of MPA was carried out using 2,4 dinitrophenylhydrazine (DNPH). The standard solution of MPA (10 mg/mL) was reacted with an excess amount of DNPH for an hour. The MPA-DNPH derivative was then injected into HPLC with detection at 375 nm [18]. The MPA-DNPH derivatives obtained from reacting 0.1 mg MPA with various concentrations (12.5, 25.0, 75.0 and 100.0 mg/mL) of DNPH solution in a 2.5 mL capped vial were used to study the effect of DNPH concentration and reaction time.

Prednisolone (P) was also tested for derivatization with DNPH and was used as I.S. There should be a single peak and no overlap with any interfering peaks in the sample. The P-DNPH derivative was obtained from the reaction of 0.10 g DNPH in 10 mL MeOH and 0.48 mL concentrate HCl with 0.05 g prednisolone [19]. The reaction mixture was placed on a heated water bath at 60oC for 30 min, allowed to cool down to room temperature and deionized water (60 mL) was added until the derivative product had completely precipitated. The derivative product was filtered through a glass filter and washed with 5 mL 2 M HCl twice, followed by 50 mL deionized water. It was then recrystallized in a mixture of chloroform and methanol prior to use.

## ii. Solid-phase extraction of MPA-DNPH derivative

220 Chromatography – The Most Versatile Method of Chemical Analysis

100 mg (Waters Associates, U.S.A.) was used.

**2.3. Method development and validation** 

mixture of chloroform and methanol prior to use.

i. Derivatization of MPA with DNPH

concentration and reaction time.

**2.2. Instruments and apparatus** 

Hospital, Khon Kaen University, Khon Kaen, Thailand.

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,

Human blood plasma samples were kindly obtained from the Blood Bank, Srinagarin

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.

Study on the hydrazine derivatization of MPA was carried out using 2,4 dinitrophenylhydrazine (DNPH). The standard solution of MPA (10 mg/mL) was reacted with an excess amount of DNPH for an hour. The MPA-DNPH derivative was then injected into HPLC with detection at 375 nm [18]. The MPA-DNPH derivatives obtained from reacting 0.1 mg MPA with various concentrations (12.5, 25.0, 75.0 and 100.0 mg/mL) of DNPH solution in a 2.5 mL capped vial were used to study the effect of DNPH

Prednisolone (P) was also tested for derivatization with DNPH and was used as I.S. There should be a single peak and no overlap with any interfering peaks in the sample. The P-DNPH derivative was obtained from the reaction of 0.10 g DNPH in 10 mL MeOH and 0.48 mL concentrate HCl with 0.05 g prednisolone [19]. The reaction mixture was placed on a heated water bath at 60oC for 30 min, allowed to cool down to room temperature and deionized water (60 mL) was added until the derivative product had completely precipitated. The derivative product was filtered through a glass filter and washed with 5 mL 2 M HCl twice, followed by 50 mL deionized water. It was then recrystallized in a 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 HPLC system.

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

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 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

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 ng/mL) of the spiked plasma standard.
