Aminolevulinic acid (5-ALA) is an endogenous, non-proteinogenic amino acid. It is a key component of the porphyrin synthesis pathway. Accurate quantitation of 5-ALA (as well as porphobilinogen) is required for the diagnosis and monitoring of acute porphyrias. Based on this Context of Use (COU), we present methodology for quantitation of 5-ALA over a curve range of 5 – 2,000 ng/mL in human plasma (NaHep). Moreover, to support the COU, the assay needed to be selective against a host of isobaric/structurally similar compounds, including Acetylalanine, hydroxyproline, creatine monohydrate, L-Leucine, L-Isoleucine, and 6-aminocaproic acid. 5-ALA is endogenously present at levels 10-15 ng/mL which determined the required LLOQ, and selection of a surrogate matrix approach (charcoal-stripped human plasma, NaHep anticoagulant). Initial efforts focused on a protein precipitation without derivitization in combination with ion pair-based reversed phase chromatography. Ultimately, the derivitization proved to be useful for several reasons, including improved LC retention (which allowed for elimination of ion-pairing reagents), and the ability to distinguish between 5-ALA and the interfering compounds based on mass difference. The extraction consists of addition of ISTD (13C5, 15N 5-ALA) followed by an acetonitrile-based protein precipitation. Following transfer and dry-down of the supernatant, samples are reconstituted in Sodium carbonate buffer and exposed to benzoyl chloride (which derivitizes the primary amine present on the 5-ALA structure). After ~1 minute of vortex-mixing, the reaction is quenched with the addition of 0.5% formic acid, and the plate is stored at 5 C prior to analysis with LC-MS. LC separations were carried out using a Shimadzu 20 series LC. Mobile phases consisted of 10 mM Ammonium formate with 0.1% formic acid (mobile phase A) and 0.1% formic acid in acetonitrile (mobile phase B). Separations were carried out using a Waters XSelect CSH column. Mass Spectrometric detection was accomplished using an API 5500 (AB Sciex) operating in MRM mode and utilizing positive mode ESI. As part of the validation, all calibrators were prepared using surrogate matrix, with all QCs prepared using native matrix, with the exception that LQC and HQC were also prepared in surrogate matrix for the purpose of establishing short and long term matrix stability in surrogate matrix. QC levels included endogenous QC, LMQC, MQC, and HQC, with LMQC, MQC, and HQC prepared by spiking additional 5-ALA into pre-characterized native matrix. Short and long term stability were established in both native and surrogate matrices. Recovery and matrix factor were evaluated in native matrix by inverting the post-spiked concentrations of unlabeled 5-ALA and stable-isotope labeled (SIL-5-ALA) (i.e. the SIL-5-ALA was spiked at theoretical in-plate LQC, MQC, and HQC levels, while unlabeled 5-ALA was spiked at theoretical in-plate ISTD concentration). Selectivity, hemolysis and selectivity were assessed by characterizing the endogenous levels of 5-ALA and then adding 2-5x the measured concentration of 5-ALA, followed by subsequent extraction and analysis. The overall goal of the validation was to demonstrate assay ruggedness in a manner consistent with the ICH-M10 draft guidelines for an assay that met the requirements of the COU. All validation tests were successfully completed.
Understand how to apply ICH M10 draft guidelines to validation of a bioanalytical method for measurement of endogenous compounds.
Understand how to apply straightforward benzoyl chloride derivatization-based extraction for measurement of 5-ALA
Understanding the context of use, be able to apply a straightforward derivatization-based extraction to differentiate 5-ALA from structurally similar compounds that may be present in study samples.