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Determination of Unbound Urinary Amino Acids Incorporated
with Creatinine Normalization by LC-MS/MS Method with
CLAM-2000 Online Sample Pre-treatment

Introduction

Free or unbound amino acids are important metabolites in 22 free amino acids and creatinine in urine samples. A
human blood and urine [1]. The pro le of unbound amino derivatization-free LC-MS/MS amino acid method [5] with
acids in urine are the reference indication of metabolic stable isotope labelled IS was employed. An on-line sample
imbalances and amino acid transport disorders as well as pre-treatment module CLAM-2000 coupled with
dietary protein adequacy and assimilation. Creatinine LC-MS/MS makes the analysis fully-automated, which
produced by muscle metabolism is excreted in the urine, enables from adding internal standards, sample and
which can be used to normalize the metabolite levels to solvent mixing, shaking for protein-crash and ltration to
compensate the large variation due to different intakes of transferring the nal sample solution to LC-MS/MS for
water and uid food [2-4]. The aim of this study is to analysis.
develop a reliable LC-MS/MS method for quantitation of

Experimental

A stock solution of 22 amino acids (AA) and creatinine sample preparation automatically for a connected
(CRE) were prepared from powder standards. Two LC-MS/MS system. The automated operation includes
isotope-labelled amino acid standards, phenylalanine adding IS, sample-solvent mixing, shaking and ltration
(ring-d5) and serine (2,3,3-d3) were added to the samples etc. The prepared sample was subsequently transferred to
as internal standard by CLAM-2000 prior to LC-MS/MS LC-MS/MS triple quadrupole system (LCMS-8040) for
analysis. A total of 28 urine samples were collected from analysis (Figure 1). An Amino Acid Column (100x3mm,
health individuals of different ages and genders and used 3µm) was adopted for separation of the 22 compounds
for amino acid analysis in this study. The CLAM-2000 is an with an optimized gradient elution program. The detailed
automated on-line sample pre-treatment module LC and MS/MS conditions are compiled in Table 1.
(Shimadzu Corporation), which was employed for urine

Automated process takes 3 to 5 minutes per sample

Sample Reagent Shaking Filtration Heating Sample Samples are transferred to
Pipetting Pipetting Transfer LC-MS/MS automatically.

Figure 1: Work ow of CLAM-2000: a fully automated sample preparation module coupled with LCMS-8040

2