Page 42 - Clinical-Medical Solution for Clinical Chemistry

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

Automated batch-run for analysis of 22 AA in 28 urine specimens

A total of 28 urine specimens were collected from 14 A batch-run includes (A) Blank, (B) Calibration series, (C)
male and 14 female health volunteers age between 20 QCs and (D) samples. The sequence starts from a blank
and 30 years old. The urine samples were stored in (milli-Q water), followed by calibration series → bank →
sealed plastic tubes at -20°C. The samples were analysed QC low and QC high → blank → 8-10 samples → blank
in a sequenced batch-run on the CLAM-2000 coupled → QCs → 8-10 samples. The blank was used to ensure
with LCMS-8040. The automated pre-treatment of each no sample carry-over, while QC low and QC high were
sample on CLAM-2000 involves: (a) pipetting 40 µL of placed between different group of samples to monitor
urine, 120 µL of organic solvent (MeOH/ACN) and 40 µL the loss during sample preparation and ensure the
of IS, (b) votexing the mixture, (c) vacuum ltering to reliability of the results. There was not signi cant matrix
obtain clear sample solution and (d) transferring to the effect of the method as observed in a separate study.
autosampler and injecting to LCMS-8040.

Total AA and Total Creatinine-Normalized AA (TCNA)
Figure 3(a) shows the results of total 22 amino acid (AA) AA / CRE, named as Total Creatinine-Normalized AA
and creatinine (CRE) of 28 urine specimens. The (TCNA). It can be seen that the 28 urine specimens can
variation of both total AA and CRE are signi cant, e.g., be classi ed into three groups according to the TCNA
the total AA scattering in 543~16,640 uM and CRE in ratios:
438~2,261 uM. Figure 3(b) displays their molar of Total

Conc. (uM)
(a)

(b)

Figure 3: (a) Total AA and creatinine (uM) and (b) Total creatinine-normalized AA (TCNA) of
28 urine specimens from 14 male and 14 female health volunteers age at 20~30 years old

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