Starts with setting up goals. #

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Elements of method development #

1. Analyte.
   • Is there isobaric compounds?
   • Analyte pKa value.
2. SPE
   • sorbent.
   • washing reagent, eluting reagent
3. Instrument.
   • LC column.
   • condition of instrument.

Elements of method validation #

1. Linearity (linear range)
2. Verify with external QC
3. Analyte recoveries at low end and high end
4. Precision (CV)

Case study #

Method Goals

1. Clean extract
2. Without the need for derivatization
3. Resolve isomeric/isobaric species

Elements of method development

1. Analyte.

Methylmalonic acid (MMA)

2. SPE

• To select a type of sorbent (Luna® Omega PS C18) for acidic compounds
• Sorbent pKa

• Equilibrating reagent: 1mL of 1% acetic acid in water;
• Washing reagent: 0.5mL of 50:50 methanol/Water;
• Eluting reagent: 2 x 0.6mL 2% ammonium hydroxide in Methanol;
• Reconstitute with 200 uL of 0.1% formic acid in water (mobile phase A)

3. Experimental conditions

Table 2: LC Conditions

Elements of method validation
same as mentioned above

phenomenex - Method Development for Underivatized MMA from Serum