Sample preparation method for determination of mycophenolic acid in plasma using ISOLUTE® SLE+

Figure 1: Structural formula of mycophenolic acid.

Introduction

Mycophenolic acid (Figure 1) is an active metabolite produced by the hydrolysis of the immunosuppressive agent Mycophenolate Mofetil in the body. It is considered important to control pharmacokinetic parameters such as AUC (area under the blood concentration-time curve) and trough concentration in order to prevent acute rejection after organ transplantation.

In this application note, ISOLUTE® SLE+ was used as a sample pretreatment cartridge for mycophenolic acid determination in plasma using the principles of supported liquid extraction (SLE). UV based methods are commonly used to measure mycophenolic acid. In this application note, we have developed a method that can be used for both UV measurement and LC/ MS/MS measurement. LC/MS/MS assays are highly selective and less susceptible to the effects of other drugs used in combination during treatment.

ISOLUTE® SLE+ Supported Liquid Extraction plates and cartridges offer an efficient alternative to traditional liquid- liquid extraction (LLE) for bioanalytical sample preparation, providing high analyte recoveries, no emulsion formation, and significantly reduced sample preparation.

Analytes

Mycophenolic acid (Mycophenolic acid, CAS: 137234-62-9)
 

Internal Standards

Indomethacin (Indomethacin, CAS: 53-86-1)

Sample Preparation Procedure

Format

ISOLUTE® SLE+ 400 µL Sample Volume Cartridges, Part number: 820-0055-B
An equivalent 96-well plate format
(Part number 820-0400-P01 is also available)

Sample Pre-Treatment

To 200 μL of plasma, add 5.0 μg/mL of indomethacin (internal standard) followed by 200 μL of 2% formic acid aqueous solution. Vortex mix for 30 seconds.

Sample Loading

Load 400 μL of the sample solution onto the cartridge and apply gentle pressure (3 psi) or vacuum (-0.2 bar) to initiate flow.
Allow the sample to absorb for at least 5 minutes and wait for samples to stabilize. Ensure that all sample solution is absorbed onto the diatomaceous earth bed.

Sample Elution

Add 900 μL of methyl tert-butyl ether (MTBE) and allow to flow under gravity for 5 minutes. Then, add another 900 μL of the solvent, and allow to stand for 5 minutes or more. If required, the extractions can be completed with application of gentle pressure (3 psi) or vacuum (-0.2 bar) (10–30 seconds).

Evaporation and Reconstitution

Evaporate the extract with a nitrogen gas evaporator and reconstitute the extract with water: methanol (1:1, v/v, 2 mL).In the case of UV measurement, the reconstituted solution should be measured as it is, or the reconstituted solution can be concentrated to a volume of less than 2 mL, depending on the sensitivity of UV measurement.

Dilution for LC/MS/MS

Additional dilution of the reconstituted assay solutions should be performed depending on the range of calibrated concentrations in the LC/MS/MS used*.
*In this application note, a 10-fold dilution was performed using water : methanol (1:1, (v/v)).

UHPLC Conditions

Instrument

Nexera LC-30AD (Shimadzu)

Cartridge

ACQUITY UPLC® BEH C18 1. 7 μm (2.1 mm × 50 mm cartridge; Waters)

Mobile Phase

A: 0.1% (v/v) Formic acid aqueous solution B: Acetonitrile

Flow Rate

0.4 mL min

Gradient Condition

Time/% of B: 0/5 →3/95 →4/95→ 4.1/5 →6.5/5

Cartridge Temperature

40 °C

Injection Volume

1 µL

Mass Spectrometry Conditions

Equipment

LCMS-8060 (Shimadzu)

Ionization Mode

ESI positive

Nebulizer Gas Flow Rate

3 L/min

Flow Rate of Drying Gas

10 L/min

Heating Gas Flow

10 L/min

Interface Temperature

350 °C

DL Temperature

200 °C

Heat Block Temperature

350 °C

CID Gas

270 kPa

SRM Transition

Mycophenolic acid: m/z 321. 00 > 207.15, Rt 2.26 min, Collision Energy;-22
Indomethacin (IS): m/z 358. 00 > 139.05, Rt 2.66 min, Collision Energy;-22

Figure 2: Calibration Curves and SRM Chromatograms of Mycophenolic Acids.

Calibration Curve

Figure 2 shows the calibration curves for mycophenolic acid and the SRM (Selected Reaction Monitoring) chromatograms.
SRM mode allows measurements that are more selective than UV-detection. The quantitation range was 0.1 to 200 ng/mL, which covers the required blood concentration range (0.5 to 20 μg/mL) for TDM (Therapeutic Drug Monitoring) of mycophenolic acid, and the multiple correlation coefficient (r2) was 0.999 or more, showing good linearity.

Confirmation of Analyte Recovery and Matrix Factors

Sample pre-treatment is very important in samples of biological origin. ISOLUTE® SLE+ supported liquid extraction cartridges were used to eliminate the effects of matrix components such as proteins, phospholipids, and salts and to allow improved quantitative analyses. Figure 3 shows the SRM chromatograms obtained by pretreatment of the control plasma spiked with 5.0 μg/mL mycophenolic acid. No interference from contaminants in the plasma was observed.

Recovery rates and matrix factors after SLE pretreatment for 3 mycophenolic acid plasma concentrations (0.5, 5.0, and 20.0 μg/mL) are shown in Table 1. Recovery was calculated by
comparing the peak area of samples spiked with mycophenolic acid pre-extraction (A) with that of blank samples spiked after extraction (B).
 
Figure 3. SRM chromatograms after ISOLUTE® SLE+ treatment with 5.0 μg/mL of mycophenolic acid in plasma.


Matrix factors were calculated by comparing the area values of (B) and the standard solution (S). As a result, a recovery rate of more than 90% was obtained at each concentration, and the values of matrix factors were sufficiently low. It was quantitatively confirmed that ISOLUTE SLE+ pretreatment effectively eliminates matrix effects.

*Recovery rate = [A]/[B] × 100 Matrix factor = 1-[B]/[S] × 100.
 

Tips and Tricks for Sample Pretreatment with ISOLUTE® SLE+

In supported liquid extraction, the charge (ionization) of the solute (analyte) should be suppressed as much as possible to facilitate the distribution of the target compound into
the organic solvent. This is especially important for highly polar compounds. Changing the pH of the sample solution improves the efficiency of extracting the sample solution using supported liquid extraction. For acidic compounds such as mycophenolic acid, acidification of the sample solution to inhibit analyte ionization can improve the transfer into organic solvents and increase extraction efficiency. Table 2 shows the changes in recovery rates due to pH differences in the sample solutions and extraction solvents.

In addition, unlike solid-phase extractions, in which the applied samples passes through the cartridge, ISOLUTE® SLE+ absorbs all the added samples into diatomaceous earth. Therefore, it is crucial that samples be processed in volumes appropriate for each cartridge size. If the volume of sample is too small to ensure adequate application, appropriate sample dilution is required to ensure an adequate volume is applied. Table 3 shows the change in the recovery rate due to the difference in the volume of sample applied.

*10 μL of the samples were diluted with 2% formic acid aqueous solution to 200 μL and applied to ISOLUTE® SLE+

Ordering Information

This application note was prepared in collaboration with the Pharmaceutical Department of Gunma University Hospital, Japan

Literature number: AN926