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Determination of ascorbic acid in biological
samples using liquid chromatography with
reference to sample preparation

• Roman Kandár, Pavla áková, Michal Richter
• University of Pardubice
• Faculty of Chemical Technology
• Department of Biological and Biochemical Sciences

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AIM

• To find suitable precipitation reagent regarding
  to stability and recovery of ascorbic acid

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Blood taking

• Plastic tubes containing gel
• Vacuette no. 455071, Greiner Labortechnik Co.,
  Austria
• With DL-Dithiothreitol (D-5545, Sigma-Aldrich
  Chemie Gmbh, Germany)
• Without DL-Dithiothreitol (D-5545, Sigma-Aldrich
  Chemie Gmbh, Germany)
• Serum samples were stored at 80 C

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Sample preparation
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Deproteination

• Metaphosphoric acid (5)
• Perchloric acid (1 mol/L)
• Trichloroacetic acid (10)
• Sulfosalicylic acid (10)
• Acetonitrile with hydrochloric acid
• Acetonitrile with acetic acid
• Methanol with hydrochloric acid
• Ethanol with hydrochloric acid

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• Centrifugation (22 000 x g 10 min 4 C)
• Filtration of supernatant through 0.20 mm nylon
  filter

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HPLC analysis

• Mobile phase
• Methanol - 25 mmol/L phosphate buffer (595,
  v/v), pH 4.75
• Flow rate
• 0.5 mL/min
• Column
• MAG-1, 4.6 x 150 mm, Biospher PSI 200 C18, 5 mm
  (Czech Republic)
• Temperature
• 25 C
• Inject
• 10 ml

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HPLC apparatus

• Pump Shimadzu LC-10ADVP
• Autosampler Shimadzu SIL-10ADVP
• Column Oven Shimadzu CTO-10ACVP
• UV-VIS Detector Shimadzu SPD-10AVP
• System Controller Shimadzu SCL-10AVP
• CSW 32 - Chromatography Station (DataApex)

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RESULTS
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Suitable protein precipitants
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Chromatogram of standard (0.0 mmol/L) MPA (5)
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Chromatogram of standard (5.1 mmol/L) MPA (5)
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Chromatogram of standard (101.1 mmol/L) MPA (5)
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Chromatogram of serum sample (67.4 mmol/L) MPA
(5)
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Chromatogram of standard (0.0 mmol/L) PCA (1
mol/L)
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Chromatogram of standard (5.1 mmol/L) PCA (1
mol/L)
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Chromatogram of standard (101.1 mmol/L) PCA (1
mol/L)
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Chromatogram of serum sample (81.0 mmol/L) PCA
(1 mol/L)
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Chromatogram of standard (0.0 mmol/L) TCA (10)
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Chromatogram of standard (5.1 mmol/L) TCA (10)
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Chromatogram of standard (101.1 mmol/L) TCA (10)
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Chromatogram of serum sample (117.2 mmol/L) TCA
(10)
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Chromatogram of standard (0.0 mmol/L) SSA (10)
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Chromatogram of standard (5.1 mmol/L) SSA (10)
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Chromatogram of standard (101.1 mmol/L) SSA (10)
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Chromatogram of serum sample (83.2 mmol/L) SSA
(10)
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• The only two protein precipitants (MPA and PCA)
  are suitable

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Stability of ascorbic acid
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• In consideration of stability of ascorbic acid is
  perchloric acid as protein precipitant unsuitable

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Long-term stability
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• Stability of ascorbic acid in serum sample (-80
  C) without DL-Dithiothreitol
• Less than a month
• Stability of ascorbic acid in serum sample (-80
  C) with DL-Dithiothreitol
• More than five years !

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Analytical performance
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• Intra-assay
• CV 2.1
• Inter-assay
• CV 5.8
• Linearity
• (2.0 250.0) mmol/L
• Limit of detection
• 25 pmol

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CONCLUSIONS

• The only two protein precipitants (metaphosphoric
  acid and perchloric acid) led to satisfactory
  recoveries of ascorbic acid.
• In consideration of stability of ascorbic acid is
  perchloric acid as protein precipitant
  unsuitable.
• Stability of ascorbic acid in serum sample (-80
  C) with DL-Dithiothreitol is more than five
  years.
• Moreover sixty ascorbic acid samples can be
  prepared at a time and placed in the autosampler
  for analysis.
• The method is suitable for the determination of
  uric acid too.

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ACKNOWLEDGEMENTS

• This work was supported by grant MSM0021627502.